Novobiocin Analogues as Anticancer Agents

ABSTRACT

Novel analogues and derivatives of novobiocin are provided, including compounds having modifications to the amide side chain, coumarin ring, and sugar moieties. The compounds of the present invention are useful as heat shock protein 90 inhibitors, and may be used as anticancer and neuroprotective agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/266,149 filed on Nov. 3, 2005, which claims the benefit of U.S.Provisional Application No. 60/624,566 filed on Nov. 3, 2004, each ofwhich is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was sponsored by the National Institutes of HealthCOBRE in Protein Structure and Function Grant No. NIH 31207, and thegovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the synthesis and identification ofnovobiocin analogues useful as a class of anticancer agents and/orneuroprotective agents. The compounds of the present invention act byinhibition of the Hsp90 protein-folding machinery.

2. Description of Related Art

The 90 kDa heat shock proteins (“Hsp90”) belong to a family ofchaperones that regulate intracellular functions and are required forthe refolding of denatured proteins following heat shock, as well as theconformational maturation of a large number of key proteins involved incellular processes. The Hsp90 family of chaperones is comprised of fourdifferent isoforms. Hsp90α (inducible/major form) and Hsp90β(constitutive/minor form) are found predominately in the cytosol, the94-kDa glucose-regulated protein (“GRP94”) is localized to theendoplasmic reticulum, and Hsp75/tumour necrosis factor receptorassociated protein 1 (“TRAP-1”) resides mainly in the mitochondrialmatrix. These Hsp90s bind to client proteins in the presence ofcochaperones, immunophilins, and partner proteins to make themultiprotein complex responsible for conformational maturation of newlyformed nascent peptides into biologically active three-dimensionalstructures.

As discussed more fully below, Hsp90 is an ATP-dependent protein with anATP binding site in the N-terminal region of the active homodimer.Disruption of the ATPase activity of Hsp90 results in thedestabilization of multiprotein complexes and subsequent ubiquitinationof the client protein, which undergoes proteasome-mediated hydrolysis.More specifically, in an ATP-dependent fashion, Hsp70 binds to newlysynthesized proteins cotranslationally and/or posttranslationally tostabilize the nascent peptide by preventing aggregation. Stabilizationof the Hsp70/polypeptide binary complex is dependent upon the binding ofHsp70 interacting protein (“HIP”), which occurs after Hsp70 binds to thenewly formed peptide. Hsp70-Hsp90 organizing protein (“HOP”) containshighly conserved tetratricopeptide repeats (“TPRs”) that are recognizedby both Hsp70 and Hsp90, promoting the union of Hsp70/HIP and Hsp90,which results in a heteroprotein complex. In the case of telomerase andsteroid hormone receptors, the client protein is transferred from theHsp70 system to the Hsp90 homodimer with concomitant release of Hsp70,HIP, and HOP. Upon binding of ATP and an immunophilin with cis/transpeptidyl prolyl-isomerase activity (FKBP51, FKBP52, or CyPA), theensemble folds the client protein into its three-dimensional structure.In a subsequent event, p23 binds Hsp90 near the N-terminal regionpromoting the hydrolysis of ATP and release of the folded protein, Hsp90partner proteins, and ADP.

Examples of proteins dependent upon Hsp90 for conformational maturationinclude oncogenic and cellular Src kinases (v-Src, Hck, Lck), Raf, p185,mutant p53 (not normal p53), telomerase, steroid hormone receptors,polo-like kinase (“PLK”), protein kinase B (“AKT”), death domain kinase(“RIP”), MET kinase, focal adhesion kinase (“FAK”), aryl hydrocarbonreceptor, RNA-dependent protein kinase (“PKR”), nitric oxide synthase(“NOS”), centrosomal proteins, PI3 kinases, androgen receptor (“AR”),matrix metalloproteinase-2 (“MMP2”) and others. In addition, otherproteins, such as cyclin dependent kinase 4 (“CDK4”), cyclin dependentkinase 6 (“CDK6”), estrogen receptor, human epidermal growth factorreceptor 2 (“Her-2” or “erbB2”) are thought to be client proteins ofHsp90. Of these Hsp90 client proteins, Raf, PLK, RIP, AKT, FAK,telomerase, HER-2, and MET kinase are directly associated with the sixhallmarks of cancer: (1) self-sufficiency in growth signals; (2)insensitivity to antigrowth signals; (3) evasion of apoptosis; (4)unlimited replication potential; (5) sustained angiogenesis; and (6)tissue invasion/metastasis. Consequently, Hsp90 is a target for thedevelopment of cancer therapeutics because multiple signaling pathwayscan be simultaneously inhibited by disruption of the Hsp90 proteinfolding machinery.

Hsp90 contains two nucleotide-binding sites: the N-terminal ATP bindingsite is the region to which geldanamycin (“GDA”),17-(allylamino)-17-demethoxygeldanamycin (“17-AAG”), herbimycin A(“HB”), and radicicol bind (see Roe et al., Structural Basis forInhibition of the Hsp90 Molecular Chaperone by the Antitumor AntibioticsRadicicol and Geldanamycin, J. Med. Chem. 1999, 42, 260-266) and theC-terminus, which was recently shown to bind novobiocin (see Marcu etal., The Heat Shock Protein 90 Antagonist Novobiocin Interacts with aPreviously Unrecognized ATP-binding Domain in the Carboxy Terminis ofthe Chaperone, J. Biol. Chem. 2000, 276, 37181).

Novobiocin

The C-terminal portion of Hsp90 is required for dimerization andrepresents a promising target for inhibitors. Unfortunately, the abilityof novobiocin to cause degradation of Hsp90 clients is relatively weak(about 700 μM in SKBr3 breast cancer cells). Thus, there remains a needto develop other Hsp90 inhibitors as useful anti-cancer agents. Mostpreferably, these new Hsp90 inhibitors have decreased toxicity,increased solubility, and/or increased selectivity for Hsp90.

It is also contemplated that the Hsp90 inhibitors of the presentinvention will be useful as neuroprotective agents. The accumulation ofprotein aggregates within or outside neurons is a common characteristicof the two most common age-related neurodegenerative diseases,Alzheimer's disease, with plaques enriched in β-amyloid peptides (“Aβ”)and neurofibrillary tangles (“NFTs”) containing hyperphsophorylated Tauprotein, and Parkinson's disease (“PD”) with Lewy bodies composedprimarily of fibrillar α-synuclein. However, even less frequent butequally debilitating nervous system diseases such as Huntington'sdisease, amyotrophic lateral sclerosis (“ALS”), prion diseases, and thetauopathies also share the characteristic of aggregated proteindeposits. A growing body of evidence now indicates that strategies thatpromote either refolding or degradation of hyperphosphorylated Tauenhance cell survival in the presence of over-expressed Tau or mutanthuman Tau. See, e.g., Shimura et al., Binding of Tau to heat shockprotein 27 leads to decreased concentration of hyperphosphorylated tauand enhanced cell survival, J. Biol. Chem., 2004, 279:17957-17962; Douet al., Chaperones increase association of Tau protein withmicrotubules, Proc. Natl. Acad. Sci. USA, 2003, 100:721-726; Kosik &Shimura, Phosphorylated tau and the neurodegenerative foldopathies,Biochim. Biophys. Acta., 2005, 1739:298-310; Shimura et al., CHIP-Hsc70complex ubiquitinates phosphorylated tau and enhances cell survival, J.Biol. Chem., 2005 279:4869-4876. Such observations suggest that thecellular machinery needed for removal of misfolded proteins may becompromised in neurodegenerative diseases.

More specifically, the interaction of Hsp90 with cochaperones thatregulate cell-specific responses to stress has led to the identificationof Hsp90 and the cochaperones Hsp70 and CHIP (carboxy-terminus of theHsp70-interacting protein) as strong candidates in determining the fateof neuronal protein aggregates. This has been most clearly demonstratedin the case of the hyperphosphorylated Tau protein in NFTs inAlzheimer's disease and the “tauopathies” due to mutations in the taugene. Low concentrations of Hsp90 inhibitors appear to up-regulateexpression of Hsp90 and co-chaperones that decrease aggregated Tau andincrease neuronal survival. However, most of the known Hsp90 inhibitorsare toxic to many cell types, limiting their potential for chronic useto delay the progression of neurodegenerative diseases. Thus, thereremains a need to develop other Hsp90 inhibitors as usefulneuroprotective agents.

Additional aspects of the invention, together with the advantages andnovel features appurtenant thereto, will be set forth in part in thedescription which follows, and in part will become apparent to thoseskilled in the art upon examination of the following, or may be learnedfrom the practice of the invention. The objects and advantages of theinvention may be realized and attained by means of the instrumentalitiesand combinations particularly pointed out in the appended claims.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to novel compounds useful as Hsp90inhibitors, and in particular as anti-cancer an neuroprotective agents.

In one aspect, the invention encompasses compounds according to FormulaI

wherein R¹ is hydrogen, alkyl, alkenyl, alkynyl, carbocyclic,heterocyclic, aryl, aralkyl, carboxyl, amido, amino, sulfanyl, sulfenyl,sulfonyl, or ether; or R¹ together with X² and the atom to which R¹ isattached form a heterocyclic ring having 4 to 8 ring members with atleast one heteroatom selected from oxygen or nitrogen; or R¹ togetherwith X⁴ and the atom to which R¹ is attached form a heterocyclic ringhaving 4 to 8 ring members with at least one heteroatom selected fromoxygen or nitrogen;

wherein R² is hydrogen, hydroxy, or —R⁵—OR⁹, wherein R⁸ is a covalentbond or alkyl, and R⁹ is C-amido or acyl; or R² together with R³ and theatoms to which they are attached form a heterocyclic ring having 4 to 8ring members with at least one heteroatom selected from oxygen ornitrogen;

wherein R³ is hydrogen, hydroxy, or —R¹⁰—O—R¹¹, wherein R¹⁰ is acovalent bond or alkyl, and R¹¹ is C-amido or acyl; or R³ together withR² and the atoms to which they are attached form a heterocyclic ringhaving 4 to 8 ring members with at least one heteroatom selected fromoxygen or nitrogen;

wherein R⁴ is hydrogen, hydroxy, carboxyl, —R¹²—O—R¹³, or —R¹²—R¹⁴; andwherein R¹² is a covalent bond or alkyl, and R¹³ is C-amido or acyl, andR¹⁴ is N-amido, —POR₁₅R¹⁶—SO₂R¹⁷, or sulfonamido, and wherein R¹⁵, R¹⁶,R¹⁷ are independently alkoxy;

wherein R⁵ is hydrogen, alkyl, alkenyl, alkynyl, aryl, or aralkyl;

wherein R⁶ is hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy,aryloxy or aralkoxy;

wherein X₁ is —O—, —CO—, or —N—;

wherein X₂ is —O—, —N—, —NR¹⁸—, —CR¹⁹—, or —CO—; and wherein R¹⁸ and R¹⁹are hydrogen, alkyl, alkenyl, or alkynyl; or X₂ together with R¹ and theatom to which R¹ is attached form a heterocyclic ring having 4 to 8 ringmembers with at least one heteroatom selected from oxygen or nitrogen;

wherein X₄ is —O—, —CR²⁰—, —CO—, or —N—, wherein R²⁰ is hydrogen, alkyl,alkenyl, alkynyl, or hydroxy; or wherein X₄ together with R¹ and theatoms to which they are attached form a heterocyclic ring having 4 to 8ring members with at least one heteroatom selected from oxygen ornitrogen;

wherein X₅, is —CR²¹— or —N—, wherein R²¹ is hydrogen, alkyl, alkenyl,or alkynyl;

wherein X₆, is —CR²²— or —N—, wherein R²² is hydrogen, alkyl, alkenyl,alkynyl, alkoxy, aryl, aralkyl, halogen, or nitro; or X₆ together withX₉ and the carbon at position 7 form a heterocylic ring having 4 to 8ring members with at least one heteroatom selected from oxygen ornitrogen;

wherein X₈, is —CR²³— or —N—, wherein R²³ is hydrogen, alkyl, alkenyl,or alkynyl;

wherein X₉ is alkyl, alkenyl, alkynyl, ether, secondary or tertiaryamino, or sulfanyl; or X₉ together with X₆ and the carbon at position 7form a heterocylic ring having 4 to 8 ring members with at least oneheteroatom selected from oxygen or nitrogen;

wherein at least one of X₁, X₂, X₄, X₅, X₆, X₈ is not —CR—; and

wherein n is 0, 1, 2, or 3.

In still another aspect, the present invention is directed to compoundsof Formula I that are coumarin compounds wherein X₁ is —O— and X₂ is—CO—.

In still another aspect, the present invention is directed to compoundsof Formula I that are isocoumarin compounds wherein X₁ is —CO— and X₂ is—O—.

In still a further aspect, the present invention is directed todes(dimethyl) derivatives and analogues of novobiocin in which R⁴ and R⁵are both hydrogen.

In still another aspect, the present invention is directed to desmethoxyderivatives and analogues of novobiocin in which R⁶ is hydrogen.

In yet another aspect, the present invention is directed to compoundsaccording to the Formula I(F):

wherein X₄, X₅, X₆, X₈, X₉, R², R³, R⁴, R⁵, R⁶, and n are defined as setforth above, and wherein R^(a), R^(b) and R^(c) are independentlyhydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, oraralkyl; and wherein R^(b) may also be oxidized to form a carbonyl.

In still another aspect, the invention encompasses compounds accordingto the Formula I(F)(i):

wherein X₉, R², R³, R⁴, R⁵, and R⁶ are defined as set forth above, andwherein R^(a) and R^(c) are independently hydrogen, alkyl, alkenyl,alkynyl, carbocyclic, heterocyclic, aryl, or aralkyl.

In still another aspect, the present invention is directed to compoundsaccording to the Formula I(F)(ii):

wherein X₄, X₅, X₆, and X₈ are defined as set forth above, and whereinR^(a), R^(b) and R^(c) are independently hydrogen, alkyl, alkenyl,alkynyl, carbocyclic, heterocyclic, aryl, or aralkyl; and wherein R^(b)may also be oxidized to form a carbonyl.

In still another aspect, the present invention is directed to compoundsencompassed by the Formula I(F)(iii):

In yet another aspect, the present invention is directed to compoundsaccording to the Formula I(G):

wherein X₅, X₆, X₈, X₉, R², R³, R⁴, R⁵, R⁶, and n are defined as setforth above, and wherein R^(a), R^(b) and R^(c) are independentlyhydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, oraralkyl; and wherein R^(b) may also be oxidized to form a carbonyl.

In a further aspect, the present invention is directed to compoundsaccording to the Formula I(G)(i):

wherein X₉, R², R³, R⁴, R⁵, R⁶ are defined as set forth above, andwherein R^(a) and R^(c) are independently hydrogen, alkyl, alkenyl,alkynyl, carbocyclic, heterocyclic, aryl, or aralkyl.

In yet another aspect, the present invention is directed to compoundsaccording to Formula I(G)(ii):

wherein X₅, X₆, and X₈ are defined as set forth above, and whereinR^(a), R^(b) and R^(c) are independently hydrogen, alkyl, alkenyl,alkynyl, carbocyclic, heterocyclic, aryl, or aralkyl; and wherein R^(b)may also be oxidized to form a carbonyl.

In still a further aspect, the present invention is directed tocompounds according to Formula I(G)(iii):

In a further aspect, the present invention is directed to compoundsaccording to Formula I(H):

wherein X₄, X₆, X₈, X₉, R¹, R², R³, R⁴, R⁵, R⁶, and n are defined as setforth above.

In still a further aspect, the invention comprises compounds accordingto the Formula I(H)(i):

wherein X₉, R¹, R², R³, R⁴, R⁵, and R⁶ are defined as set forth above.

In still another aspect, the present invention is directed to compoundsaccording to Formula I in which R² and R³ form a cyclic carbonate.

In still another aspect, the present invention is directed to compoundsaccording to Formula I in which the sugar ring is modified to include adiol at R² and R³.

In still another aspect, the present invention is directed to compoundsaccording to Formula I in which sugar is modified to include a2′-carbamate at R².

In still another aspect, the present invention is directed to thecompounds of Formula I in which the coumarin ring is modified to includea lower alkoxy or nitro substitution at the 6-position of the coumarinring.

In a further aspect, the present invention encompasses compoundsaccording to Formula I(J):

wherein X₁, X₂, X₄, X₅, X₈, R¹, R², R³, R⁴, R⁵, R⁶, and n are defined asset forth above; and wherein R^(a) and R^(b) are independently hydrogen,alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, or aralkyl;and wherein R^(b) may also be oxidized to form a carbonyl.

In still a further aspect, the present invention is directed tocompounds according Formula I(J)(i):

wherein X₁, X₂, X₄, X₅, X₈, R¹, R², R³, R⁴, R⁵, R⁶, n, and R^(a) aredefined as set forth above.

In still a further aspect, the present invention is directed tocompounds according to the Formula I(J)(ii):

wherein X₁, X₂, X₄, X₅, X₈, R¹, R², R³, R⁴, R⁵, R⁶, n, and R^(a) aredefined as set forth above.

In still another aspect, the present invention is directed to compoundsencompassed by Formula I(K):

wherein X₄, X₅, X₆, X₈, X₉, R¹, R², R³, R⁴, R⁵, R⁶, and n are defined asset forth above; and wherein R¹⁸ is hydrogen, alkyl, alkenyl, oralkynyl.

In yet another aspect, the present invention is directed to compoundsencompassed by Formula I(K)(i):

wherein X₉, R¹, R², R³, R⁴, R⁵, and R⁶ are defined as set forth above;and wherein R¹⁸ is hydrogen, alkyl, alkenyl, or alkynyl.

In still a further aspect, the present invention is directed to4-deshydroxy derivatives and analogues of novobiocin in which X₄ is—CR²⁰— and R²⁰ is hydrogen.

In yet a further aspect, the present invention is directed to8-desmethyl derivatives and analogues of novobiocin in which X₈ is—CR²²— and R²² is hydrogen.

In still another aspect, the present invention encompasses compoundsaccording to the Formula I(L):

wherein X₄, X₅, X₆, X₈, X₉, R¹, R², R³, R⁴, R⁵, R⁶, and n are defined asset forth above.

In yet another aspect, the present invention is directed to compoundsaccording Formula I(L)(i):

wherein X₉, R¹, R², R³, R⁴, R⁵, R⁶, and n are defined as set forthabove.

In still another aspect of the present invention, the novobiocinderivatives and analogues of the present invention are modified so thatthe sugar is modified as set forth below:

In still another aspect, the present invention is directed to dimers ofthe foregoing compounds. In particular, exemplary dimers are provided bythe formula:

wherein X is alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic orheterocyclic; and wherein R², R³, R⁴, R⁵, and R⁶ are set forth above.

In another aspect, the present invention comprises the in which thelinker is a heterocylic pyrrole as shown below:

It is contemplated that one or more compounds of the present inventionwill be useful for inhibiting heat-shock protein 90 activity byadministering one of more of the compounds of the present invention to acell or subject and observing a decrease in the expression of aheat-shock protein 90 client protein.

According to another aspect, the present invention provides apharmaceutical composition, which comprises a therapeutically-effectiveamount of one or more compounds of the present invention or apharmaceutically-acceptable salt, ester or prodrug thereof, togetherwith a pharmaceutically-acceptable diluent or carrier.

Additional aspects of the invention, together with the advantages andnovel features appurtenant thereto, will be set forth in part in thedescription which follows, and in part will become apparent to thoseskilled in the art upon examination of the following, or may be learnedfrom the practice of the invention. The objects and advantages of theinvention may be realized and attained by means of the instrumentalitiesand combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative ratios of phospho-AKT by Western blot analyseswhen the compounds of Example 1 were tested for their ability to inhibitHsp90 in Skbr3 breast cancer cells. Total protein concentration of eachlysate was determined and equal amounts of protein were run in each laneof the gels. For the graphs shown in FIG. 1, the O.D.'s (opticaldensity) of the Western bands for phospho-AKT were measured, as were theO.D.'s for actin probed as controls on the same blots. To obtain thegraphed values, all specific O.D.'s (for Hsp90 clients) were normalizedto the respective actin O.D.

FIG. 2 is a western blot analysis of Skbr3 cells treated with novobiocinanalogue denominated herein as KU-3/A2 (2′-carbamate) and KU-1/A4 (diol)for 24 hours. After incubation, the cells were harvested, lysed, andequal amounts of the protein lysates loaded into SDS wells. Afterelectrophoresis, the gel was probed with Her-2 and actin (control)antibodies. The specific decrease in Her-2 levels is a result of Hsp90inhibition that leads to Her-2 degradation.

FIG. 3 (top panel) is a western blot analysis of prostate cancer LNCaPcells treated with KU-1/A4. The bottom panel is a western blot analysisof prostate cancer LAPC-4 cells incubated with KU-1/A4. Actin was usedas a control in both assays.

FIG. 4 shows the dose dependent effects of KU-1/A4 on Aβ-induced celldeath in primary neurons. The compound was added two hours before the βAand the viability was determined at 48 hours. The data representsstandard error of the means (“S.E.M.”) from about 1500 cells from 3preparations. #, p<0.0001 for control vs. Aβ only. **, p<0.001. Aβ onlyvs. Aβ+KU-1/A4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

Molecular terms, when used in this application, have their commonmeaning unless otherwise specified. It should be noted that thealphabetical letters used in the formulas of the present inventionshould be interpreted as the functional groups, moieties, orsubstitutents as defined herein. Unless otherwise defined, the symbolswill have their ordinary and customary meaning to those skilled in theart.

The term “acyl” refers to —COR wherein R used in this definition ishydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocylic, aryl, oraralkyl. Most preferably, R is hydrogen, alkyl, aryl, or aralkyl.

The term “amido” indicates either a C-amido group such as —CONR′R″ or anN-amido group such as —NR′COR″ wherein R′ and R″ as used in thisdefinition are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy,carbocyclic, heterocylic, aryl, or aralkyl. A “sulfoamido” groupincludes the —NR′—SO₂—R″. Most preferably, R′ and R″ are hydrogen,alkyl, aryl, or aralkyl.

The term “amino” signifies a primary, secondary or tertiary amino groupof the formula —NR′R″ wherein R′ and R″ as used in this definition areindependently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, carbocyclic,heterocyclic, aralkyl, or other amino (in the case of hydrazide) or R′and R″ together with the nitrogen atom to which they are attached, forma ring having 4-8 atoms. Thus, the term “amino”, as used herein,includes unsubstituted, monosubstituted (e.g., monoalkylamino ormonoarylamino), and disubstituted (e.g., dialkylamino or aralkylamino)amino groups. Amino groups include —NH₂, methylamino, ethylamino,dimethylamino, diethylamino, methyl-ethylamino, pyrrolidin-1-yl orpiperidino, morpholino, etc. Other exemplary “amino” groups forming aring include pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,indolyl, indazolyl, purinyl, quinolizinyl. The ring containing the aminogroup may be optionally substituted with another amino, alkyl, alkenyl,alkynyl, halo, or hydroxyl group.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred“alkyl” groups herein contain 1 to 12 carbon atoms. Most preferred are“lower alkyl” which refer to an alkyl group of one to six, morepreferably one to four, carbon atoms. The alkyl group may be optionallysubstituted with an amino, alkyl, halo, or hydroxyl group.

The term “alkoxy” denotes oxy-containing groups substituted with analkyl, or cycloalkyl group. Examples include, without limitation,methoxy, ethoxy, tert-butoxy, and cyclohexyloxy. Most preferred are“lower alkoxy” groups having one to six carbon atoms. Examples of suchgroups include methoxy, ethoxy, propoxy, butoxy, isopropoxy, andtert-butoxy groups.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double bond or triple bondrespectively.

The term “aryl” means a carbocyclic aromatic system containing one, twoor three rings wherein such rings may be attached together in a pendantmanner or may be fused. The term “fused” means that a second ring ispresent (i.e., attached or formed) by having two adjacent atoms incommon (i.e., shared) with the first ring. The term “fused” isequivalent to the term “condensed.” The term “aryl” embraces aromaticgroups such as phenyl, naphthyl, tetrahydronaphthyl, indane, andbiphenyl. The aryl group may optionally be substituted with an amino,alkyl, halo, hydroxyl, carbocyclic, heterocyclic, or another aryl group.

The term “aralkyl” embraces aryl-substituted alkyl moieties. Preferablearalkyl groups are “lower aralkyl” groups having aryl groups attached toalkyl groups having one to six carbon atoms. Examples of such groupsinclude benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, anddiphenylethyl. The terms benzyl and phenylmethyl are interchangeable.

The term “aryloxy” embraces aryl groups, as defined above, attached toan oxygen atom. The aryloxy groups may optionally be substituted with ahalo, hydroxyl, or alkyl group. Examples of such groups include phenoxy,4-chloro-3-ethylphenoxy, 4-chloro-3-methylphenoxy,3-chloro-4-ethylphenoxy, 3,4-dichlorophenoxy, 4-methylphenoxy,3-trifluoromethoxyphenoxy, 3-trifluoromethylphenoxy, 4-fluorophenoxy,3,4-dimethylphenoxy, 5-bromo-2-fluorophenoxy, 4-bromo-3-fluorophenoxy,4-fluoro-3-methylphenoxy, 5,6,7,8-tetrahydronaphthyloxy,3-isopropylphenoxy, 3-cyclopropylphenoxy, 3-ethylphenoxy,4-tert-butylphenoxy, 3-pentafluoroethylphenoxy, and3-(1,1,2,2-tetrafluoroethoxy)phenoxy.

The term “aralkoxy” embraces oxy-containing aralkyl groups attachedthrough an oxygen atom to other groups. “Lower aralkoxy” groups arethose phenyl groups attached to lower alkoxy group as described above.Examples of such groups include benzyloxy, 1-phenylethoxy,3-trifluoromethoxybenzyloxy, 3-trifluoromethylbenzyloxy,3,5-difluorobenzyloxy, 3-bromobenzyloxy, 4-propylbenzyloxy,2-fluoro-3-trifluoromethylbenzyloxy, and 2-phenylethoxy.

The term “carboxyl” refers to —R′C(══O)OR″, wherein R′ and R″ as used inthis definition are independently hydrogen, alkyl, alkenyl, alkynyl,carbocyclic, heterocylic, aryl, or aralkyl or R′ can additionally be acovalent bond. “Carboxyl” includes both carboxylic acids, and carboxylicacid esters. The term “carboxylic acid” refers to a carboxyl group inwhich R″ is hydrogen. Such acids include formic, acetic, propionic,butyric, valeric acid, 2-methyl propionic acid, oxirane-carboxylic acid,and cyclopropane carboxylic acid. The term “carboxylic acid ester” or“ester” refers to a carboxyl group in which R″ is alkyl, alkenyl,alkynyl, carbocyclic, heterocylic, aryl, or aralkyl.

The term “carbocyclic” refers to a group that contains one or morecovalently closed ring structures, and that the atoms forming thebackbone of the ring are all carbon atoms. The ring structure may besaturated or unsaturated. The term thus distinguishes carbocyclic fromheterocyclic rings in which the ring backbone contains at least onenon-carbon atom. The term carbocylic encompasses cycloalkyl ringsystems.

The terms “cycloalkane” or “cyclic alkane” or “cycloalkyl” refer to acarbocyclic group in which the ring is a cyclic aliphatic hydrocarbon,for example, a cyclic alkyl group preferably with 3 to 12 ring carbons.“Cycloalkyl” includes, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, and the like. Thecycloalkyl group may be optionally substituted with an amino, alkyl,halo, or hydroxyl group.

The term “ether” refers to the group —R′—O—R″ wherein R′ and R″ as usedin this definition are independently hydrogen, alkyl, alkenyl, alkynyl,carbocyclic, heterocylic, aryl, or aralkyl, and R′ can additionally be acovalent bond attached to a carbon.

The terms “halo” or “halogen” refer to fluoro, chloro, bromo or iodo,usually regarding halo substitution for a hydrogen atom in an organiccompound.

The term “heterocyclic or heterocycle” means an optionally substituted,saturated or unsaturated, aromatic or non-aromatic cyclic hydrocarbongroup with 4 to about 12 carbon atoms, preferably about 5 to about 6,wherein 1 to about 4 carbon atoms are replaced by nitrogen, oxygen orsulfur. Exemplary heterocyclic which are aromatic include groupspyridinyl, furanyl, benzofuranyl, isobenzofuranyl, pyrrolyl, thienyl,1,2,3-triazolyl, 1,2,4-triazolyl, indolyl, imidazolyl, thiazolyl,thiadiazolyl, pyrimidinyl, oxazolyl, triazinyl, and tetrazolyl.Exemplary heterocycles include benzimidazole, dihydrothiophene, dioxin,dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan,indole, 3-H indazole, 3-H-indole, imidazole, indolizine, isoindole,isothiazole, isoxazole, morpholine, oxazole, oxadiazole, oxathiazole,oxathiazolidine, oxazine, oxadiazine, piperazine, piperidine, purine,pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyrimidine, pyridazine,pyrrole, pyrrolidine, tetrahydrofuran, tetrazine, thiadiazine,thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine,thiophene, thiopyran, triazine, and triazole. The heterocycle may beoptionally substituted with an amino, alkyl, alkenyl, alkynyl, halo,hydroxyl, carbocyclic, thio, other heterocyclic, or aryl group.Exemplary heterocyclic groups include 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 1-indolyl, 2-indolyl, 3-indolyl, 1-pyridyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 1-imidazolyl, 2-imidazolyl, 3-imidazolyl,4-imidazolyl, 1-pyrazolyl, 2 pyrazolyl, 3-pyrazolyl, 4-pyrazolyl,5-pyrazolyl, 1-pyrazinyl, 2-pyrazinyl, 1-pyrimidinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 1-pyridazinyl, 2-pyridazinyl,3-pyridazinyl, 4-pyridizinyl, 1-indolizinyl, 2-indolizinyl,3-indolizinyl, 4-indolizinyl, 5-indolizinyl, 6-indolizinyl,7-indolizinyl, 8-indolizinyl, 1-isoindolyl, 2-isoindolyl, 3-isoindolyl,4-isoindolyl, 5-isoindolyl.

The term “hydroxy” or “hydroxyl” refers to the substituent —OH.

The term “oxo” shall refer to the substituent ═O.

The term “nitro” means —NO₂.

The term “sulfanyl” refers to —SR′ where R′ as used in this definitionis hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocylic, aryl, oraralkyl.

The term “sulfenyl” refers to —SOR′ where R′ as used is this definitionis hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocylic, aryl, oraralkyl.

The term “sulfonyl” refers to —SOR′ where R′ as used in this definitionis hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, heterocylic, aryl, oraralkyl.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. “Optionally” is inclusive of embodiments in which thedescribed conditions is present and embodiments in which the describedcondition is not present. For example, “optionally substituted phenyl”means that the phenyl may or may not be substituted, and that thedescription includes both unsubstituted phenyl and phenyl wherein thereis substitution. “Optionally” is inclusive of embodiments in which thedescribed conditions is present and embodiments in which the describedcondition is not present.

The compounds of the present invention can exist in tautomeric,geometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-geometric isomers, E- andZ-geometric isomers, R— and S— enantiomers, diastereomers, d-isomers,1-isomers, the racemic mixtures thereof and other mixtures thereof, asfalling within the scope of the invention.

Also included in the family of compounds of the present invention arethe pharmaceutically acceptable salts, esters, and prodrugs thereof. Theterm “pharmaceutically-acceptable salts” embraces salts commonly used toform alkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Suitable pharmaceutically acceptable acidaddition salts of compounds of the present invention be prepared frominorganic acid or from an organic acid. Examples of such inorganic acidsare hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric,and phosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic, stearic,cyclohexylaminosulfonic, algenic, galacturonic acid. Suitablepharmaceutically-acceptable base addition salts of compounds of thepresent invention include metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethylenediamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocain. All of these salts may be prepared by conventional means fromthe corresponding compounds of by reacting, for example, the appropriateacid or base with the compounds of the present invention.

As used herein, the term “pharmaceutically acceptable ester” refers toesters which hydrolyze in vivo and include, but are not limited to,those that break down readily in the human body to leave the parentcompound or a salt thereof. Suitable ester groups include, for example,those derived from pharmaceutically acceptable aliphatic carboxylicacids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioicacids, in which each alkyl or alkenyl moiety advantageously has not morethan 6 carbon atoms. Examples of particular esters include formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, commensurate with areasonable risk/benefit ratio, and effective for their intended use,where possible, of the compounds of the invention. The term “prodrug”refers to compounds that are rapidly transformed in vivo to yield theparent compound of the above formulae, for example, by hydrolysis inblood. A thorough discussion is provided in T. Higuchi and V. Stella,Prodrugs as Novel delivery Systems, Vol. 14 of the A.C.S. SymposiumSeries and in Edward B. Roche, ed., Bioreversible Carriers in DrugDesign, American Pharmaceutical Association and Pergamon Press, 1987,both of which are incorporated by reference herein.

According to another aspect, the present invention provides apharmaceutical composition, which comprises a therapeutically-effectiveamount of one or more compounds of the present invention or apharmaceutically-acceptable salt, ester or prodrug thereof, togetherwith a pharmaceutically-acceptable diluent or carrier.

The compositions may be formulated for any route of administration, inparticular for oral, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal administration. The compositions may beformulated in any conventional form, for example, as tablets, capsules,caplets, solutions, suspensions, dispersions, syrups, sprays, gels,suppositories, patches and emulsions.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject lonidamineanalogue or derivative from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich may serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations.

The “patient” or “subject” to be treated with the compounds of thepresent invention can be any animal, and is preferably a mammal, such asa domesticated animal or a livestock animal. More preferably, thepatient is a human.

The term “inhibit” or “inhibiting” refers to a statistically significantand measurable reduction in activity, preferably a reduction of at leastabout 10% versus control, more preferably a reduction of about 50% ormore, still more preferably a reduction of about 80% or more.

A “therapeutically effective amount” is an amount of a compound of thepresent invention or a combination of two or more such compounds, whichinhibits, totally or partially, the progression of the condition oralleviates, at least partially, one or more symptoms of the condition. Atherapeutically effective amount can also be an amount that isprophylactically effective. The amount that is therapeutically effectivewill depend upon the patient's size and gender, the condition to betreated, the severity of the condition and the result sought. For agiven patient and condition, a therapeutically effective amount can bedetermined by methods known to those of skill in the art. For example,in reference to the treatment of cancer using the compounds of thepresent invention, a therapeutically effective amount refers to thatamount which has the effect of (1) reducing the size of the tumor, (2)inhibiting (that is, slowing to some extent, preferably stopping) tumormetastasis, (3) inhibiting to some extent (that is, slowing to someextent, preferably stopping) tumor growth, and/or, (4) relieving to someextent (or, preferably, eliminating) one or more symptoms associatedwith the cancer.

Several of the compounds of the present invention have been shown toinhibit Hsp90 in vitro. As such, it is contemplated that therapeuticallyeffective amounts of the compounds of the present invention will beuseful as anti-cancer agents and/or neuroprotective agents.

In the context of cancer and neuroprotection, it is contemplated thatsome of the compounds of the present invention may be used with otherHsp90 inhibitors, chemotherapeutic agents, and/or neuroprotectiveagents.

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. Those skilled in theart will readily understand that known variations of the conditions andprocesses of the following preparative procedures can be used to preparethese compounds.

Example 1 Synthesis of Novobiocin Analogues

In an effort to increase the affinity of novobiocin for the C-terminalATP binding site, a library of novobiocin analogue compounds thatcontained both modified coumarin and sugar derivatives was prepared. Thecompounds were prepared as set forth in the scheme below along with aprocedure recently developed for the synthesis of noviose. See Yu etal., Synthesis of (−)-Noviose from2,3-O-Isopropylidene-D-erythronolactol, J. Org. Chem. 2004, 69,7375-7378, which is incorporated by reference.

The novobiocin analogues prepared according to the scheme includedmodification of the coumarin ring by shortening of the amide side chainand removal of the 4-hydroxy substituent (A) (see Madhavan et al., NovelCoumarin Derivatives of Heterocyclic Compounds as Lipid Lowering Agents,Bioorg. Med. Chem. Lett. 2003, 13, 2547, which is incorporated byreference), removal of both the 4-hydroxy and amide linker (B), stericreplacements of both the 4-hydroxy and benzamide ring (C), and1,2-positional isomers of the noviosyl linkage (D and E).

These selected coumarin rings were coupled with trichloroacetimidate ofnoviose carbonate in the presence of boron trifluoride etherate as shownin scheme below. See Shen et al., Syntheses of Photolabile NovobiocinAnalogues, Bioorg. Med. Chem. Lett. 2004, 14, 5903. The resulting cycliccarbonates (A1-E1) were treated with methanolic ammonia to provide2′-carbamoyl (A2-E2), 3′-carbamoyl (A3-E3), and descarbamoyl products(A4-E4) in good yields. See also Yu et al., Hsp90 Inhibitors Identifiedfrom a Library of Novobiocin Analogues, J. Am. Chem. Soc. 2005, 127,12778-12779, which is incorporated by reference.

wherein R¹ in the above scheme is hydrogen, amido, amino, or aryl; and

wherein R² in the above scheme is hydrogen, alkyl, or hydroxyl.

Overall, the following twenty-three (23) analogues of novobiocin wereprepared, which are set forth below:

N-(7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-2-oxo-2H-chromen-3-yl)acetamide(A1). Noviose carbonate trichloroacetimidate (180 mg, 0.50 mmol) and7-hydroxy-3-acetamino-coumarin A (133 mg, 0.60 mmol) were dissolved inCH₂Cl₂ (7 mL) before boron trifluoride etherate (30 μL, 0.03 mmol) wasadded to the suspension at 25° C. The mixture was stirred at 25° C. for8 h and quenched with Et₃N (0.4 mL, 2.8 mmol). The solvent was removedand the residue purified by chromatography (SiO₂, 5% acetone in CH₂Cl₂)to afford A1 (134 mg, 64%) as a colorless solid: [α]²⁵ _(D)=−71.0° (c,0.1, CH₂Cl₂); ¹H NMR (CD₃Cl 400 MHz) δ 8.67 (s, 1H), 8.00 (br s, 1H),7.46 (d, J=8.6 Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 7.00 (dd, J=2.3, 8.6 Hz,1H), 5.82 (d, J=1.5 Hz, 1H), 5.02 (dd, J=1.5, 7.8 Hz, 1H), 4.94 (t,J=7.8 Hz, 1H), 3.62 (s, 3H), 3.30 (d, J=7.8 Hz, 1H), 2.26 (s, 3H), 1.37(s, 3H), 1.21 (s, 3H); ¹³C NMR (CD₃Cl 100 MHz) δ 169.7, 159.2, 157.4,153.5, 151.4, 129.2, 123.9, 122.8, 115.1, 114.6, 104.1, 94.7, 83.4,78.3, 77.6, 77.5, 61.1, 27.9, 25.2, 22.4; IR (film) ν_(max) 1819, 1764,1615, 1560, 1507, 1375, 1300, 1212, 1168, 1107, 1072, 1034, 1002, 969cm⁻¹, HRMS (FAB⁺) m/z 420.1285 (M+H⁺, C₂₀H₂₂NO₉ requires 420.1294).

(2R,3R,4R,5R)-2-(3-acetamido-2-oxo-2H-chromen-7-yloxy)-4-hydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-3-ylcarbamate (A2),(3R,4S,5R,6R)-6-(3-acetamido-2-oxo-2H-chromen-7-yloxy)-5-hydroxy-3-methoxy-2,2-dimethyltetrahydro-2H-pyran-4-yl carbamate (A3) andN-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-2-oxo-2H-chromen-3-yl)acetamide(A4). Noviosylated coumarin A1 (20 mg, 0.047 mmol) was dissolved inmethanolic ammonia (7.0 M, 2 mL) at 25° C. and stirred for 24 h. Thesolvent was evaporated and the residue purified by preparative HPLC(SiO₂, 20% 2-propanol in hexanes) to afford A2 (4.2 mg, 22%), A3 (8.6mg, 42%) and A4 (3.5 mg, 20%) as colorless solids.

A2: [α]²⁵ _(D)=−143.2° (c, 0.11, 50% MeOH in CH₂Cl₂); ¹HNMR (50% CD₃ODin CD₂Cl₂ 400 MHz) δ 8.58 (s, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.01 (s, 1H),6.97 (d, J=8.4 Hz, 1H), 5.59 (d, J=2.0 Hz, 1H), 5.03 (dd, J=2.0, 3.6 Hz,1H), 4.25 (dd, J=3.6, 9.7 Hz, 1H), 3.57 (s, 3H), 3.30 (d, J=9.7 Hz, 1H),2.19 (s, 3H), 1.31 (s, 3H), 1.13 (s, 3H); ¹³CNMR (50% CD₃OD in CD₂Cl₂100 MHZ) δ 168.8, 157.2, 156.4, 155.5, 149.5, 126.9, 122.9, 120.4,112.6, 112.3, 101.6, 94.8, 82.5, 77.0, 71.9, 64.7, 59.9, 27.0, 22.1,20.6; IR (film) ν_(max) 3473, 1716, 1689, 1610, 1540, 1528, 1505, 1375,1240, cm⁻¹; HRMS (FAB⁺) m/z 437.1565 (M+H⁺, C₂₀H₂₅N₂O₉ requires437.1560).

A3: [α]²⁵ _(D)=−116.2° (c, 0.24, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 8.59 (s, 1H), 7.52 (d, J=10.8 Hz, 1H), 7.04 (s, 1H), 7.03 (d,J=10.8 Hz, 1H), 5.56 (d, J=2.4 Hz, 1H), 5.25 (dd, J=3.2, 9.8 Hz, 1H),4.20 (dd, J=2.4, 3.2 Hz, 1H), 3.58 (s, 3H), 3.35 (d, J=9.8 Hz, 1H), 2.22(s, 3H), 1.27 (s, 3H), 1.18 (s, 3H); ¹³CNMR (CD₃OD 100 MHZ) δ 171.6,158.8, 158.7, 158.1, 151.8, 128.9, 125.6, 122.5, 114.4, 114.2, 103.1,99.1, 81.6, 79.0, 71.8, 69.7, 60.1, 27.9, 22.9, 22.4; IR (film) ν_(max)3470, 1716, 1686, 1615, 1538, 1523, 1505, 1372, 1242, 1120 cm⁻¹; HRMS(FAB⁺) m/z 437.1576 (M+H⁺, C₂₀H₂₅N₂O₉ requires 437.1560).

A4: [α]²⁵ _(D)=−351.6° (c, 0.06, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 8.58 (s, 1H), 7.51 (d, J=8.3 Hz, 1H), 7.03 (s, 1H), 7.02 (d,J=8.3 Hz, 1H), 5.55 (d, J=2.3 Hz, 1H), 4.10 (dd, J=3.3, 9.6 Hz, 1H),4.03 (dd, J=2.4, 3.3 Hz, 1H), 3.60 (s, 3H), 3.38 (d, J=9.6 Hz, 1H), 2.21(s, 3H), 1.30 (s, 3H), 1.13 (s, 3H); ¹³CNMR (CD₃OD 100 MHZ) δ 171.6,158.9, 158.8, 151.8, 128.9, 125.7, 122.5, 114.3, 114.1, 103.1, 99.2,84.2, 78.8, 71.5, 68.4, 61.1, 28.2, 22.9, 22.4; IR (film) ν_(max) 3326,1714, 1674, 1613, 1558, 1553, 1108 cm⁻¹; HRMS (FAB⁺) m/z 394.1492 (M+H⁺,C₁₉H₂₄O₈ requires 394.1502).

7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-2H-chromen-2-one(B1). Noviose carbonate trichloroacetimidate (90 mg, 0.25 mmol) and7-hydroxy-coumarin B (48 mg, 0.30 mmol) were dissolved in CH₂Cl₂ (2 mL)before boron trifluoride etherate (10 μL, 0.01 mmol) was added to thesuspension at 25° C. The mixture was stirred at 25° C. for 8 h andquenched with Et₃N (0.1 mL, 0.7 mmol). The solvent was removed and theresidue purified by chromatography (SiO₂, 2% acetone in CH₂Cl₂) toafford BI (66 mg, 73%) as a colorless solid: [α]²⁵ _(D)=−85.6° (c, 1.15,CH₂Cl₂); ¹HNMR (CDCl₃ 400 MHz) δ 7.69 (d, J=9.5 Hz, 1H), 7.43 (d, J=8.6Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 6.95 (dd, J=2.3, 8.6 Hz, 1H), 6.34 (d,J=9.5 Hz, 1H), 5.84 (d, J=1.3 Hz, 1H), 5.03 (dd, J=1.3, 7.7 Hz, 1H),4.94 (t, J=7.7 Hz, 1H), 3.62 (s, 3H), 3.30 (d, J=7.7 Hz, 1H), 1.37 (s,3H), 1.20 (s, 3H); ¹³CNMR (CDCl₃ 100 MHZ) δ 161.2, 158.9, 155.9, 153.5,143.5, 129.4, 114.7, 114.4, 113.7, 104.4, 94.6, 83.4, 78.3, 77.8, 77.5,61.0, 27.9, 22.4; IR (film) ν_(max) 1809, 1730, 1612, 1171, 1157, 1109cm⁻¹; HRMS (FAB⁺) m/z 363.1083 (M+H⁺, C₁₈H₁₉O₈ requires 363.1080).

(3R,4S,5R,6R)-5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-4-ylcarbamate (B2),(2R,3R,4R,5R)-4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-3-ylcarbamate (B3) and7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-2H-chromen-2-one(B4). Noviosylated coumarin B1 (25 mg, 0.07 mmol) was dissolved inmethanolic ammonia (7.0 M, 2 mL) at 25° C. and stirred for 24 h. Thesolvent was evaporated and the residue purified by preparative TLC(SiO₂, 25% acetone in methylene chloride) to afford B2 (4.3 mg, 16%), B3(14.5 mg, 52%) and B4 (4.0 mg, 17%) as colorless solids.

B2: [α]²⁵ _(D)=−85.1° (c, 0.71, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.91 (d, J=9.5 Hz, 1H), 7.58 (dd, J=1.3, 9.0 Hz, 1H), 7.04 (s,1H), 7.03 (d, J=9.0 Hz, 1H), 6.30 (d, J=9.5 Hz, 1H), 5.65 (d, J=2.1 Hz,1H), 5.04 (dd, J=2.6, 3.4 Hz, 1H), 4.28 (dd, J=3.4, 9.9 Hz, 1H), 3.62(s, 3H), 3.39 (d, J=9.5 Hz, 1H), 1.35 (s, 3H), 1.15 (s, 3H); ¹³CNMR(CD₃OD 100 MHZ) δ 161.7, 159.7, 157.5, 155.3, 144.1, 129.1, 113.6,113.4, 112.8, 103.0, 96.4, 83.9, 78.5, 73.4, 66.2, 60.8, 28.0, 21.8; IR(film) ν_(max) 3438, 2982, 2932, 1731, 1616, 1403, 1338, 1280, 1117,1002, 963 cm⁻¹; HRMS (FAB⁺) m/z 380.1333 (M+H⁺, C₁₇H₂₁O₇ requires380.1345).

B3: [α]²⁵ _(D)=−111.8° (c, 0.18, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.91 (d, J=9.5 Hz, 1H), 7.58 (d, J=8.3 Hz, 1H), 7.05 (s, 1H),7.04 (d, J=8.3 Hz, 1H), 6.30 (d, J=9.9 Hz, 1H), 5.59 (d, J=2.4 Hz, 1H),5.25 (dd, J=3.2, 9.8 Hz, 1H), 4.20 (dd, J=2.4, 3.2 Hz, 1H), 3.59 (d,J=9.5 Hz, 1H), 3.57 (s, 3H), 1.36 (s, 3H), 1.17 (s, 3H); ¹³CNMR (CD₃OD100 MHZ) δ 161.7, 159.9, 157.7, 155.3, 144.2, 129.1, 113.6, 113.5,112.7, 102.9, 98.6, 81.1, 78.6, 71.4, 69.3, 60.6, 27.5, 22.0; IR (film)ν_(max) 3359, 2979, 2937, 1710, 1615, 1317, 1120, 1092, 995 cm⁻¹; HRMS(FAB⁺) m/z 380.1327 (M+H⁺, C₁₇H₂₁O₇ requires 380.1345).

B4: [α]²⁵ _(D)=−129.4° (c, 0.18, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.91 (d, J=9.5 Hz, 1H), 7.57 (dd, J=2.4, 10.4 Hz, 1H), 7.02 (m,2H), 6.27 (dd, J=4.5, 9.5 Hz, 1H), 5.57 (d, J=2.4 Hz, 1H), 4.11 (dd,J=3.3, 9.5 Hz, 1H), 4.03 (dd, J=2.4, 3.3 Hz, 1H), 3.60 (s, 3H), 3.39 (d,J=9.5 Hz, 1H), 1.35 (s, 3H), 1.12 (s, 3H); ¹³CNMR (CD₃OD 100 MHZ) δ161.7, 160.9, 155.4, 144.2, 129.0, 113.5, 113.4, 112.6, 102.9, 98.8,83.7, 78.4, 71.1, 67.9, 60.7, 27.7, 22.0; IR (film) ν_(max) 3415, 2984,2934, 1730, 1718, 1707, 1615, 1118, 999, 957 cm⁻¹; HRMS (FAB⁺) m/z337.11279 (M+H⁺, C₁₇H₂₁O₇ requires 337.1287).

7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-4-methyl-3-phenyl-2H-chromen-2-one(C1). Noviose carbonate trichloroacetimidate (90 mg, 0.25 mmol) and7-hydroxy-4-methyl-3-phenyl-coumarin C (76 mg, 0.30 mmol) were dissolvedin CH₂Cl₂ (2 mL) before boron trifluoride etherate (10 μL, 0.01 mmol)was added to the suspension at 25° C. The mixture was stirred at 25° C.for 8 h and quenched with Et₃N (0.1 mL, 0.7 mmol). The solvent wasremoved and the residue purified by chromatography (SiO₂, 1% acetone inCH₂Cl₂) to afford C1 (92 mg, 73%) as a colorless solid:

[α]²⁵ _(D)=−75.8° (c, 1.41, CH₂C12); ¹HNMR (CDCl₃ 400 MHz) δ 7.80 (d,J=9.6 Hz, 1H), 7.44 (m, 3H), 7.33 (m, 2H), 7.09 (d, J=2.4 Hz, 1H), 7.01(dd, J=2.4, 5.2 Hz, 1H), 5.84 (d, J=1.3 Hz, 1H), 5.03 (dd, J=1.3, 7.7Hz, 1H), 4.94 (t, J=7.7 Hz, 1H), 3.62 (s, 3H), 3.30 (d, J=7.7 Hz, 1H),2.31 (s, 3H), 1.37 (s, 3H), 1.20 (s, 3H); ¹³CNMR (CDCl₃ 100 MHZ) δ161.0, 158.0, 153.9, 153.0, 147.4, 134.3, 130.0 (2C), 128.3 (2C), 128.0,126.2, 125.2, 115.6, 113.0, 103.7, 94.1, 82.9, 77.8, 76.7, 76.5, 60.5,27.4, 22.0, 16.5; IR (film) ν_(max) 1874, 1715, 1612, 1564, 1507, 1383,1262, 1167, 1130, 1113, 1070, 1033, 1006, 968, 936 cm⁻¹; HRMS (FAB⁺) m/z453.1554 (M+H⁺, C₂₅H₂₅O₈ requires 453.1549).

(3R,4S,5R,6R)-5-hydroxy-3-methoxy-2,2-dimethyl-6-(4-methyl-2-oxo-3-phenyl-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-4-ylcarbamate (C2),(2R,3R,4R,5R)-4-hydroxy-5-methoxy-6,6-dimethyl-2-(4-methyl-2-oxo-3-phenylyl-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-3-ylcarbamate (C3) and7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-4-methyl-3-phenyl-2H-chromen-2-one(C4). Noviosylated coumarin C1 (25 mg, 0.055 mmol) was dissolved inmethanolic ammonia (7.0 M, 2 mL) at 25° C. and stirred for 24 h. Thesolvent was evaporated and the residue purified by preparative TLC(SiO₂, 25% acetone in methylene chloride) to afford C2 (6.3 mg, 25%), C3(13.7 mg, 53%) and C4 (3.0 mg, 13%) as colorless solids.

C2: [α]²⁵ _(D)=−72.9° (c, 0.19, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.80 (d, J=9.0 Hz, 1H), 7.43 (m, 3H), 7.32 (m, 2H), 7.10 (m, 2H),5.69 (d, J=1.8 Hz, 1H), 5.06 (dd, J=2.1, 3.2 Hz, 1H), 4.30 (dd, J=3.2,9.7 Hz, 1H), 3.63 (s, 3H), 3.40 (d, J=9.7 Hz, 1H), 2.31 (s, 3H), 1.36(s, 3H), 1.18 (s, 3H); ¹³CNMR (CD₃OD 100 MHZ) δ 162.2, 159.7, 158.0,154.2, 149.2, 135.1, 130.3 (2C), 128.4 (2C), 128.1, 127.0, 124.7, 115.3,113.7, 103.2, 96.8, 84.4, 78.9, 73.8, 66.7, 61.3, 28.4, 22.3, 15.8; IR(film) ν_(max) 3474, 2986, 2924, 1713, 1605, 1382, 1355, 1263, 1124,1001, 967 cm⁻¹; HRMS (FAB⁺) m/z 470.1821 (M+H⁺, C₂₅H₂₈NO₈ requires470.1815).

C3: [α]²⁵ _(D)=−92.3° (c, 0.28, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.75 (d, J=9.5 Hz, 1H), 7.45 (m, 3H), 7.34 (m, 2H), 7.06 (m, 2H),5.63 (d, J=2.4 Hz, 1H), 5.18 (dd, J=3.2, 9.6 Hz, 1H), 4.18 (dd, J=2.4,3.2 Hz, 1H), 3.54 (s, 3H), 3.40 (d, J=9.5 Hz, 1H), 2.27 (s, 3H), 1.35(s, 3H), 1.16 (s, 3H); ¹³CNMR (CD₃CN 125 MHZ) δ 160.7, 159.0, 156.0,153.8, 148.0, 135.2, 130.1 (2C), 128.1 (2C), 127.7, 126.7, 124.4, 114.9,113.1, 103.1, 98.2, 81.0, 78.4, 71.3, 69.0, 60.7, 27.7, 22.4, 15.8; IR(film) ν_(max) 3459, 3331, 2981, 2925, 1714, 1606, 1379, 1335, 1263,1124, 1072 cm⁻¹; HRMS (FAB⁺) m/z 470.1811 (M+H⁺, C₂₅H₂₈NO₈ requires470.1815).

C4: [α]²⁵ _(D)=−86.0° (c, 0.12, 50% MeOH in CH₂Cl₂); ¹HNMR (CD₃OD 400MHz) δ 7.80 (d, J=9.6 Hz, 1H), 7.44 (m, 3H), 7.33 (m, 2H), 7.09 (m, 2H),5.60 (d, J=1.9 Hz, 1H), 4.12 (dd, J=3.3, 9.5 Hz, 1H), 4.05 (dd, J=2.4,3.1 Hz, 1H), 3.61 (s, 3H), 3.40 (d, J=9.5 Hz, 1H), 2.32 (s, 3H), 1.37(s, 3H), 1.15 (s, 3H); ¹³CNMR (CD₃OD 100 MHZ) δ 161.9, 159.6, 153.8,149.1, 134.7, 129.9 (2C), 127.9 (2C), 127.7, 126.5, 124.1, 114.7, 113.4,102.7, 98.8, 83.8, 78.4, 71.1, 68.0, 60.7, 27.8, 22.0, 15.4; IR (film)ν_(max) 3403, 2977, 2924, 1717, 1607, 1558, 1505, 1381, 1260, 1124, 992cm⁻¹; HRMS (FAB⁺) m/z 427.1750 (M+H⁺, C₂₄H₂₇O₇ requires 427.1757).

8-(7-Methoxy-6,6-dimethyl-2-oxo-tetrahydro-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-chromen-2-one(D1) Noviose carbonate trichloroacetimidate (176 mg, 0.49 mmol) and8-hydroxy-coumarin D (95 mg, 0.59 mmol) were dissolved in CH₂Cl₂ (5 mL).Boron trifluoride etherate (20 μL, 0.08 mmol) was added to thesuspension at 25° C. The resulting slurry was stirred at 25° C. for 10 hbefore the solvent was removed and the residue purified bychromatography (SiO₂, 1% MeOH in CHCl₃) to afford D1 (85 mg, 40%) as acolorless solid:

[α]_(D) ³¹=−57° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CDCl₃, 500 MHz) δ7.69 (d, J=9.6 Hz, 1H), 7.31 (t, J=9.1 Hz, 1H), 7.23 (dd, J=2.8 Hz, 9.0Hz, 1H), 7.16 (d, J=2.8 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 5.77 (d, J=1.0Hz, 1H), 5.03 (dd, J=1.2 Hz, 7.8 Hz, 1H), 4.95 (t, J=7.7 Hz, 1H), 3.62(s, 3H), 3.30 (d, J=7.7 Hz, 1H), 1.37 (s, 3H), 1.20 (s, 3H); ¹³C NMR(CDCl₃, 125 MHz) δ 160.6, 153.1, 152.1, 149.4, 142.9, 120.8, 119.3,118.0, 117.4, 113.3, 94.5, 82.9, 77.9, 77.2, 76.5, 60.5, 27.5, 22.0; IR(film) ν_(max) 3054, 2987, 1817, 1730, 1572, 1422, 1166, 1112, 1040,896, 739 cm⁻¹; HRMS (FAB⁺) m/z 363.1088 (M+H⁺, C₁₈H₁₉O₈ requires m/z363.1080).

Carbamic acid4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-8-yloxy)-tetrahydro-pyran-3-ylester (D2), carbamic acid5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-8-yloxy)-tetrahydro-pyran-4-ylester (D3),8-(3,4-Dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-pyran-2-yloxy)-chromen-2-one(D4) D1 (17 mg, 0.047 mmol) was dissolved in methanolic ammonia (2.0 M,5 mL, 10 mmol) at 25° C. and stirred for 5 h before the solvent wasremoved. The residue was purified by preparative TLC (SiO₂, 25% acetonein CH₂Cl₂) to afford D2 (3.8 mg, 21%), D3 (5.5 mg, 31%), and D4 (7.2 mg,46%) as colorless solids.

D2: [α]_(D) ³¹=−19° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CD₃OD inCD₂Cl₂, 500 MHz) δ 7.79 (d, J=9.6 Hz, 1H), 7.26 (m, 3H), 6.43 (d, J=9.6Hz, 1H), 5.59 (d, J=2.0 Hz, 1H), 5.05 (dd, J=2.1 Hz, 3.4 Hz, 1H), 4.28(m, 2H), 3.61 (s, 3H), 3.32 (m, 1H), 1.34 (s, 3H), 1.18 (s, 3H); ¹³C NMR(CD₃OD in CDCl₃, 100 MHz) δ 162.1, 158.0, 153.6, 149.3, 144.6, 121.1,119.9, 117.7, 116.6, 113.6, 97.1, 84.5, 78.8, 74.1, 66.7, 61.4, 28.6,22.4; IR (film) ν_(max) 3054, 2987, 1729, 1422, 896, 739, 705 cm⁻¹; HRMS(ESI⁺) m/z 380.1356 (M+H⁺, C₁₈H₂₂NO₈ requires m/z 380.1345).

D3: [α]_(D) ³¹=−69° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CD₃OD inCD₂Cl₂, 500 MHz) δ 7.84 (d, J=9.6 Hz, 1H), 7.30 (m, 3H), 6.44 (d, J=9.5Hz, 1H), 5.51 (d, J=2.3 Hz, 1H), 5.28 (dd, J=3.2 Hz, 9.8 Hz, 1H), 4.21(m, 1H), 3.56 (s, 1H), 3.55 (s, 3H), 1.35 (s, 3H), 1.20 (s, 3H); ¹³C NMR(CD₃OD in CDCl₃, 125 MHz) δ 161.8, 157.4, 153.3, 148.7, 144.2, 120.8,119.3, 117.4, 116.2, 113.2, 98.9, 81.3, 78.6, 71.5, 69.5, 60.8, 27.9,22.3; IR (film) ν_(max) 3054, 2987, 1732, 1422, 896, 742 cm⁻¹; HRMS(ESI⁺) m/z 380.1348 (M+H⁺, C₁₈H₂₂NO₈ requires m/z 380.1345).

D4: [α]_(D) ³¹=−91° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CD₃OD inCD₂Cl₂, 500 MHz) δ 7.82 (d, J=9.5 Hz, 1H), 7.26 (m, 3H), 6.43 (d, J=9.5Hz, 1H), 5.50 (d, J=2.3 Hz, 1H), 4.12 (dd, J=3.4 Hz, 9.3 Hz, 1H), 4.05(d, J=2.4 Hz, 1H), 3.59 (s, 3H), 3.33 (m, 1H), 1.35 (s, 3H), 1.15 (s,3H); ¹³C NMR (CD₃OD in CDCl₃, 125 MHz) δ 161.7, 153.4, 148.6, 144.2,120.7, 119.3, 117.3, 116.1, 113.1, 98.9, 83.8, 78.3, 71.1, 68.0, 60.9,28.0, 22.2; IR (film) ν_(max) 3455, 3053, 2988, 1704, 1568, 1112, 738cm⁻¹; HRMS (FAB⁺) m/z 337.1267 (M+H⁺, C₁₇H₂₁O₇ requires m/z 337.1287).

6-(7-Methoxy-6,6-dimethyl-2-oxo-tetrahydro-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-chromen-2-one(E1) Noviose carbonate trichloroacetimidate (150 mg, 0.42 mmol) and6-hydroxycoumarin E (67 mg, 0.42 mmol) were dissolved in CH₂Cl₂ (4 mL).Boron trifluoride etherate (20 μL, 0.06 mmol) was added to thesuspension at 25° C. The resulting slurry was stirred at 25° C. for 10 hbefore the solvent was removed and the residue purified bychromatography (SiO₂, 1% MeOH in CHCl₃) to afford E1 (63 mg, 42%) as acolorless solid:

[α]_(D) ³¹=−59° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CDCl₃, 500 MHz) δ7.69 (d, J=9.6 Hz, 1H), 7.30 (d, J=9.0 Hz, 1H), 7.23 (dd, J=2.7 Hz, 9.0Hz, 1H), 7.16 (d, J=2.7 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 5.77 (m, 1H),5.02 (dd, J=1.0 Hz, 7.8 Hz, 1H), 4.95 (d, J=7.7 Hz, 1H), 3.61 (s, 3H),3.30 (d, J=7.7 Hz, 1H), 1.37 (s, 3H), 1.23 (s, 3H); ¹³C NMR (CDCl₃, 125MHz) δ 160.6, 153.1, 152.1, 149.4, 142.9, 120.8, 119.3, 118.0, 117.4,113.3, 94.5, 82.9, 77.9, 77.2, 76.5, 60.5, 27.5, 22.0; IR (film) ν_(max)3054, 2987, 1818, 1730, 1422, 896, 739, 705 cm⁻¹; HRMS (FAB⁺) m/z363.1109 (M+H⁺, C₁₈H₁₉O₈ requires m/z 363.1080).

Carbamic acid5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-6-yloxy)-tetrahydro-pyran-4-ylester (E2), Carbamic acid4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-6-yloxy)-tetrahydro-pyran-3-ylester (E3),6-(3,4-Dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-pyran-2-yloxy)-chromen-2-one(E4) E1 (17 mg, 0.047 mmol) was dissolved in methanolic ammonia (7.0 M,5 mL, 35 mmol) at 25° C. and stirred for 5 h before the solvent wasremoved. The residue was purified by preparative TLC (SiO₂, 25% acetonein CH₂Cl₂) to afford compound E2 (7.8 mg, 34%), E3 (9.9 mg, 43%), and E4(4.7 mg, 23%) as colorless solids.

E2: [α]_(D) ³¹=−45° (c=0.1, 50% MeOH in CH₂Cl₂). ¹H NMR (CD₃OD inCD₂Cl₂, 500 MHz) δ 7.82 (d, J=9.6 Hz, 1H), 7.27 (m, 3H), 6.44 (d, J=9.5Hz, 1H), 5.60 (d, J=2.0 Hz, 1H), 5.05 (dd, J=2.0 Hz, 3.4 Hz, 1H), 4.28(m, 1H), 3.61 (s, 3H), 3.32 (m, 1H), 1.34 (s, 3H), 1.18 (s, 3H); ¹³C NMR(CD₃OD in CD₂Cl₂, 125 MHz) δ 161.6, 157.2, 153.1, 148.8, 143.9, 120.8,119.3, 117.4, 116.4, 113.2, 96.7, 84.1, 78.4, 73.7, 66.3, 61.3, 28.4,22.2; IR (film) ν_(max) 3054, 2987, 1729, 1422, 896, 738, 705 cm⁻¹; HRMS(ESI⁺) m/z 380.1327 (M+H⁺, C₁₈H₂₂NO₈ requires m/z 380.1345).

E3: [α]_(D) ³¹=−80° (c=0.1, 50% MeOH in CH₂Cl₂); ¹H NMR (CD₃OD inCD₂Cl₂, 500 MHz) δ 7.79 (d, J=9.5 Hz, 1H), 7.28 (d, J=2.3 Hz, 2H), 7.25(s, 1H), 6.43 (d, J=9.5 Hz, 1H), 5.50 (d, J=2.3 Hz, 1H), 5.26 (dd, J=3.2Hz, 9.8 Hz, 1H), 4.21 (t, J=2.7 Hz, 1H), 3.56 (m, 1H), 3.55 (s, 3H),1.35 (s, 3H), 1.19 (s, 3H); ¹³C NMR (CD₃OD in CD₂Cl₂, 125 MHz) δ 159.5,155.0, 151.1, 146.8, 141.8, 118.7, 117.3, 115.4, 114.4, 111.2, 96.7,79.3, 76.6, 69.6, 67.4, 59.0, 26.0, 20.4; IR (film) ν_(max) 3054, 2987,1731, 1422, 1265, 896, 742 cm⁻¹; HRMS (ESI⁺) m/z 380.1324 (M+H⁺,C₁₈H₂₂NO₈ requires m/z 380.1345).

E4: [α]_(D) ³¹=−89° (c=0.05, 50% MeOH in CH₂Cl₂); ¹H NMR (CD₃OD inCD₂Cl₂, 400 MHz) δ 7.83 (d, J=9.6 Hz, 1H), 7.26 (m, 3H), 6.44 (d, J=9.5Hz, 1H), 5.50 (d, J=2.3 Hz, 1H), 4.12 (dd, J=3.4 Hz, 9.3 Hz, 1H), 4.05(d, J=2.4 Hz, 1H), 3.59 (s, 3H), 3.33 (m, 1H), 1.34 (s, 3H), 1.14 (s,3H); ¹³C NMR (CD₃OD in CD₂Cl₂, 125 MHz) δ 162.1, 153.8, 149.2, 144.5,121.3, 119.8, 117.8, 116.8, 113.5, 99.3, 84.4, 78.8, 71.6, 68.5, 61.6,28.6, 22.8; IR (film) ν_(max) 3454, 3054, 2987, 1705, 1568, 1422, 1111,896, 738 cm⁻¹; HRMS (FAB⁺) m/z 337.1275 (M+H⁺, C₁₇H₂₁O₇ requires m/z337.1287).

As discussed more fully below, these compounds were then tested forbiological activity with respect to Hsp90 inhibition. Based on theresults, various additional modifications to the side chains at R¹ andR² in the above scheme are proposed, as well as modifications to thecoumarin ring and sugar moiety.

Example 2 Degradation of Phospho-AKT

Inhibition of Hsp90 results in the degradation of Hsp90-dependentclients via ubiquitination of the unfolded client followed byproteasome-mediated hydrolysis. To test whether Hsp90 client proteinswere degraded in the presence of these novobiocin analogues, each memberof the library from Example 1 was incubated with SKBr3 breast cancercells at a concentration of 100 μM. Western blot analysis of the proteinlysates demonstrated that several of the compounds were capable ofcausing the degradation of the Hsp90-dependent oncogenic client protein,phospho-AKT as represented in FIG. 1. Phospho-AKT was chosen as a clientprotein for this assay because of previous reports indicating thatphospho-AKT is a more sensitive indicator of Hsp90 inhibition than AKT.Geldanamycin (GDA, 0.5 μM) was used as a positive control for Hsp90inhibition.

As can be seen from FIG. 1, A4/KU-1 (diol) and A3/KU-2 (3′-carbamate)were the most potent novobiocin analogues identified, based on theirability to inhibit Hsp90 and cause the degradation of phosphorylatedAKT. As shown in FIG. 1, the most active compound identified in thisassay was A4/KU-1 from the scheme above, which contains an N-acetyl sidechain in lieu of the benzamide, lacks the 4-hydroxyl of the coumarinmoiety, and has an unmodified diol. Structure-activity relationships forthese compounds suggests that attachment of the noviose moiety to the7-position of the coumarin ring is preferred for biological activity (Bvs. D and E). Further, incorporation of the amide linker (A) resulted ingreater inhibitory activity than the unsubstituted derivative, B. It islikely that the diol (4) mimics the ribose ring in the normal substrate(ATP) and may explain why replacement with a cyclic carbonate (1) or2′-carbamate (2) resulted in decrease of activity.

Example 3 Degradation of Her-2

The IC₅₀ for Hsp90 inhibitors is sometimes determined as theconcentration of inhibitor required to produce 50% degradation of Her-2,another therapeutically important Hsp90 client protein involved inbreast cancer. When KU-1/A4 was incubated with Skbr3 breast cancer cellsat concentrations of 100 nM, 1 μM and 10 μM, a rapid decrease in Her-2was observed between 100 nM and 1 μM, as shown in the Western blot ofFIG. 2. These data are normalized against actin, a non-Hsp90 clientprotein, used as a control for non-specific degradation. These datasuggest the IC₅₀ of KU-1/A4 is in the low micromolar range, whereasnovobiocin in the same assay produces an IC₅₀ of 700 μM.

Example 4 Prostate Cancer

The steroid hormone receptors are also dependent upon the Hsp90 proteinfolding machinery for activation and hormone binding. To determinewhether KU-1/A4 had similar effects on the androgen receptor, KU-1/A4was tested in both a mutated androgen receptor-dependent prostate cancercell line (LNCaP) and a wild type androgen receptor prostate cancer cellline (LAPC-4). More specifically, the prostate cancer cells were grownin RPMI with 10% fetal calf serum in a standard fashion. Once the cellshad reached near confluence, they were treated with vehicle (DMSO) orvarying concentrations of KU-1/A4 ranging from 10 nm to 100 μM for 24hours. Cells were harvested and cell lysates prepared. Western blotanalysis was then performed on the cell lysate utilizing commerciallyavailable antibodies against the androgen receptor, AKT, HIF-1α, Her2,and Hsp90. Actin was used as the control. More specifically, WesternBlot analysis protein concentrations in serum samples were determined bythe Pierce BCA protein assay kit according to the manufacturer'sprotocol. Western blot analysis (100 mg total protein/lane to start) waselectrophoresed under reducing conditions on a SDS-PAGE gel. Theseparated proteins were transferred to a polyvinylidene difluoridemembrane (Millipore, Bedford, Mass.) for 40 minutes at 80 V. Themembranes were blocked for two hours at room temperature inTris-buffered saline (pH 7.5) containing 0.2% I-block (Tropix, Bedford,Mass.), 1% milk, and 0.1% Tween-20 (TBS-T). The membranes weresubsequently be incubated with a primary antibody to the above mentionedproteins (all of which have commercially available antibodies) overnightat 4° C. The next day the membrane was washed three times in TBS-Tfollowed by one hour incubation with an appropriate horseradishperoxidase labeled secondary antibody in blocking buffer (TBS-T). Themembranes were again washed in TBS-T and Tris-buffered saline anddeveloped in SuperSignal West Pico Chemiluminescent Substrate (Pierce,Rockford, Ill.) according to manufacturer's instructions. The blots werevisualized by exposing the enhanced chemiluminescence-reacted blot toX-ray film.

As can be seen in FIG. 3, KU-1/A4 had a dramatic effect on theconcentrations of the mutant androgen receptor, AKT, and HIF-1α at about1 μM in the LNCaP cell line. In addition, KU-1/A4 drastically reducedlevels of the androgen receptor at lower concentrations in the wild typeandrogen receptor prostate cancer cell line (LAPC-4). To verify thatKU-1/A4 was not affecting other transcriptional or translationalprocesses that could account for decreased protein, Hsp90 levels weredetermined. Under normal conditions, Hsp90 binds heat shock factor 1(HSF1), but in the presence of Hsp90 inhibitors this interaction is lostand HSF1 is able to induce the expression of Hsp90. As can be seen inFIG. 3, Hsp90 levels are significantly increased in a manner dependenton the concentration of KU-1/A4 consistent with similar resultspreviously obtained by incubation with geldanamycin and radicicol. Bothof these data are in contrast to actin, which is not an Hsp90 clientprotein and thus remains unaffected by Hsp90 inhibitors.

Prophetic Example 4 Amide Side Chain Modifications

Since KU-1/A4 was shown to be the most potent C-terminal inhibitor ofHsp90 identified in Example 1, additional derivatives of the KU-1/A4scaffold will be prepared. Modifications of the amide side chain willallow for an in depth study of the hydrophobic cavity that binds to thisportion of KU-1/A4 and the analogous benzamide of novobiocin. As such,analogues of KU-1/A4 that have increasingly larger hydrophobic groups bythe use of different commercially available or readily synthesizedanhydrides, such as those anhydrides shown in the scheme below. SeeKhoo, L. E., Synthesis of Substituted 3-Aminocoumarins from EthylN-2-Hydroxyarylideneglycinates, Syn. Comm. 1999, 29, 2533-2538, which isincorporated by reference.

wherein in the scheme R is hydrogen, alkyl, alkenyl, alkynyl, aryl,carbocylic, heterocyclic, aryl, or aralkyl, (and most preferably R ishydrogen, alkyl, aryl, and aralkyl);

and wherein R′ is hydrogen or CONH₂.

As part of this example, the amide linkage will also be reversed todetermine the optimal profile of this functionality. As set forth in thescheme below, the 7-hydroxy-3-ethyl ester coumarin will be hydrolyzed toafford the corresponding acid, which will be coupled with amines thatmimic the same side chains used in the KU-1/A4 amide studies for directcomparison of biological activity. Once coupled, the free phenols willbe noviosylated as described earlier to afford the cyclic carbonateproducts. Treatment of the carbonate with methanolic ammonia will givethe diol, 2- and 3-carbamoyl products as shown in the scheme below. SeeShen et al., Synthesis of Photolabile Novobiocin Analogues, Bioorg. Med.Chem. Lett. 2004, 14, 5903-5906, which is incorporated by reference.

wherein in the scheme R is hydrogen, alkyl, alkenyl, alkynyl, aryl,carbocylic, heterocyclic, aryl, or aralkyl;

and wherein R′ is hydrogen or CONH₂.

Most preferably, the R in the amide side chain is hydrogen alkyl, aryl,and alkaryl, and the amines used in the above scheme are NH₃,methylamine, ethylamine, propylamine, n-butylamine, and phenylamine.However, it will be appreciated to those skilled in the art that otherderivatives can be prepared in accordance with the above scheme, inaddition to the KU-1/A4 analogues shown. That is, the amide side chain,coumarin ring, and sugar may be modified in accordance with the otherexamples shown herein.

Prophetic Example 5 Isocoumarin Derivatives

To determine the most favorable interaction of the coumarin lactone withHsp90, the isocoumarin derivative of the compounds of the presentinvention will be prepared. For example, with respect to KU-1/A4, theisocoumarin will be prepared from the 4-benzyloxylactone shown in thescheme below. Treatment of the lactone with sodium cyanide, followed byHCl/pyridine is known to produce similar isocoumarins. See Wells et al.,Facile synthesis of 3-acylaminoisocoumarins, J. Org. Chem. 1971, 36,1503-1506, which is incorporated by reference. Acylation of the aminefollowed by removal of the benzyl-protecting group will provide thephenol, which will be coupled with noviose trichloroacetimidate toafford the cyclic carbonate precursor. Ammoniaolysis of the cycliccarbonate will afford both the diol and 3′-carbamoyl products.

It will be appreciated to those skilled in the art that otherisocoumarin derivatives can be prepared in accordance with the abovescheme, in addition to the KU-1/A4 analogue shown. That is, the amideside chain, coumarin ring, and sugar may be modified in accordance withthe other examples shown herein.

Prophetic Example 6 Des(Dimethyl) and Desmethoxy Sugar Analogues

Modifications to the gem-dimethyl groups and the methyl ether on thenoviose moiety will be prepared. In this example, the des(dimethyl) anddesmethoxy sugar analogues will be prepared. Using KU-/1/A4 as anexample in the scheme below, 2,3-O-isopropylidene-L-erythronolactol willbe converted to the corresponding alkene by Wittig olefination.Dihydroxylation will afford the syn diol as noted in the earliersynthesis of noviose. See Yu et al., Synthesis of (−)-Noviose from2,3-O-Isopropylidene-D-erythronolactol, J. Org. Chem. 2004, 69,7375-7378. Protection of the primary alcohol, followed by alkylation ofthe secondary alcohol will afford the orthogonally protected molecule.Selective removal of the benzyl group and oxidation of the resultantalcohol will give the aldehyde. Treatment of this aldehyde with aqueoussulfuric acid will remove the acid-labile protecting groups whilesimultaneously promoting cyclization. Id.

Similarly, the desmethoxy compound will be prepared from theappropriately functionalized lactone (Stewart et al., 2-Deoxy-L-Ribosefrom an L-Arabino-1,5-lactone, Tetrahedron Assym. 2002, 13, 2667-2672)by the addition of excess methyl Grignard to provide the primary andtertiary alcohol product. Oxidation of the primary alcohol will give thelactone, which will be reduced to the lactol before deprotection withaqueous sulfuric acid to yield the desmethoxy product. Once obtained,these sugars will be treated with carbonyl diimidazole to furnish thecyclic carbonates before coupling with the coumarin phenol. This set ofconditions is based on previous work towards the preparation ofnovobiocin photoaffinity probes. See Shen et al., Synthesis ofPhotolabile Novobiocin Analogues, Bioorg. Med. Chem. Lett. 2004, 14,5903-5906.

wherein preferably R is lower alkyl; and

wherein R′ is preferably hydrogen or —CONH₂.

It will be appreciated that other demethylated an/or dealkoxylatedderivatives can be prepared in accordance with the above scheme, inaddition to the modified KU-1/A4 derivative shown above. That is, theamide side chain, coumarin ring, and sugar may be modified in accordancewith the other examples shown herein.

Prophetic Example 7 Modified Novobiocin Derivatives

This example involves the modification to of the compounds of thepresent invention to complement the hydrogen bonding capabilities of thenucleotide bases (adenine and guanine) with those of the coumarin ringsystem as shown below. As an example, these analogues containconformationally restricted hydrogen bond donors/acceptors of KU-1/A4 (Fand G) and strategically placed hydrogen bond acceptors/donors tocomplement those found in guanine (H-L). In all cases, the hydrophobicpocket that accommodates the m-substituted benzamide ring of novobiocinwill be probed by alteration of the side chain constituents. Althoughthe schemes below are directed to preparing modifications of KU-1/A4, itwill be appreciated to those skilled in the art, that the samemodifications could be made in conjunction with other analoguesdescribed herein, such as the A-E compounds of Example 1.

Example 7F Heterocyclic Modifications to Quinolone

In this example, the coumarin ring will be modified to create Fanalogues that resemble guanine and contain a conformationally biasedhydrogen-bond donor/acceptor. The synthesis begins with commerciallyavailable 4-hydroxy-2-nitrobenzaldehyde following the procedure ofMeanwell, et al., Inhibitors of Blood Platelet cAMP Phosphodiesterase.2. Structure-Activity Relationships Associated with1,3-Dihydro-2H-imidazo[4,5-b]quinolin-2-ones Substituted withFunctionalized Side Chains, J. Med. Chem. 1992, 35, 2672-2687. Thephenol will be protected as the benzylether, followed by treatment withhydantoin phosphonate to give the corresponding olefin. See Meanwell etal., Diethyl 2,4-dioxoimidazolidine-5-phosphonate: A Wadsworth-EmmonsReagent for the Mild and Efficient Preparation of C-5 UnsaturatedHydantoins, J. Org. Chem. 1991, 56, 6897-6904. Reduction of thebenzylether, nitro, and olefin functionalities will provide theappropriate amine for subsequent addition to the carbonyl upon treatmentwith iodine. Meanwell et al., Inhibitors of Blood Platelet cAMPPhosphodiesterase, Structure-Activity Relationships Associated with1,3-Dihydro-2H-imidazo[4,5-b]quinolin-2-ones Substituted withFunctionalized Side Chains, J. Med. Chem. 1992, 35, 2672-2687. Asdepicted earlier, the unmasked phenol will be coupled with thetrichloroacetimidate of noviose carbonate, followed by removal of thecarbonate moiety to furnish analogue F.

It will be readily appreciated to those skilled in the art that theforegoing scheme for the F analogues can be readily modified to preparethe following compounds, in addition to the oxidized imidazole attachedto the quinolone shown above, by using commercially available or readilysynthesized bases. Thus, the present invention encompasses novobiocinderivatives according to the formula:

wherein X₄, X₅ X₆ X₈ are preferably each —CH—; and

wherein R^(a), R^(b), and R^(c) are independently hydrogen, alkyl,alkenyl, alkynyl, carbocyclic, heterocylic, aryl, or aralkyl; or whereinR^(b) is oxided to form the carbonyl according to the formula:

Example 7G Heterocyclic Modifications

In this example, coumarin G will be prepared from7-benzyloxy-4-hydroxy-3-nitrocoumarin, according to the scheme below.See Buckle et al., Aryloxyalkyloxy- and aralkyloxy-4-hydroxy-3-nitrocoumarins which inhibit histamine release in the rat and also antagonizethe effects of a slow reacting substance of anaphylaxis, J. Med. Chem.1979, 22, 158-168. Treatment of the 4-hydroxyl group with phosphorousoxychloride (POCl₃) will afford the corresponding 4-amino derivativeupon subsequent exposure to ammonia. See Rassochandran et al., Mildmethod for the preparation of 4-chloro-3-nitro coumarins, Indian. J.Chem. 1986, 25B, 328-329. Reduction of the nitro group, followed byreaction with triethyl orthoformate in the presence of acid will affordthe desired compound. See Trkovnik et al., Synthesis of newheterocyclocoumarins from 3,4-diamino- and 4-chloro-3-nitrocoumarins,Prep. Proced. Int. 1987, 19, 450-455. Treatment of this 3,4-diamine withother commercially or readily available orthoesters (see McElvain etal., Ketene acetals. XVI. Phenylketene diethyl- and dimethylacetals fromthe pyrolysis of the corresponding orthoesters. J. Am. Chem. Soc. 1946,68, 1917-1921) will provide a direct method for exploration of thehydrophobic pocket surrounding this moiety. The orthoesters readilycondense with 1,2-diamines to produce the corresponding heterocyliccompounds. Once prepared, these compounds will be coupled with noviosecarbonate in analogous fashion to that shown in above to afford thecorresponding G analogues of KU-1/A4.

It will be readily appreciated to those skilled in the art that theforegoing scheme can be readily modified to prepare the followingcompounds, in addition to the imidazole shown above by using differentorthoesters.

wherein X₅ X₆ X₈ are preferably each —CH—; and

wherein R^(a), R^(b), and R^(c) are independently hydrogen, alkyl,alkenyl, alkynyl, carbocyclic, heterocylic, aryl, or aralkyl; or whereinR^(b) is oxided to form the carbonyl according to the formula:

Example 7H

The nitrogen-containing H variants of the coumarin ring will be preparedfrom 2-methyl-3,5-pyridinediol, by bromination of the benzylic methylgroup, followed by hydrolysis and oxidation to the correspondingaldehyde as set forth in the scheme below. See Morisawa et al.,Anticoccidal agents. IV. Modification at the 5-position of4-deoxypyridoxol and α4-norpyridoxol, Agric. Biol. Chem. 1975, 39,1275-1281. Using conditions previously employed for the syntheses ofother coumarin derivatives by us, the aldehyde will be treated withglycine under basic conditions to yield the azacoumarin ring system. SeeBilleret et al., Convenient synthesis of 5-azacoumarins, J. Hetero.Chem. 1993, 30, 671-674. Acylation of the amine with various anhydrideswill furnish the acylated 7-hydroxyl and 4-amino derivatives, of whichthe 7-phenolic ester can be readily cleaved by subsequent treatment withpotassium carbonate in methanol. The resulting phenol will be coupledwith noviose carbonate as described earlier.

While the scheme above illustrates the modified coumarin of KU-1/A4 witha limited number of amide side chain substitutions, it will beappreciated to those skilled in the art that other derivatives can beprepared in accordance with the above scheme, in addition to the KU-1/A4analogues shown. That is, the amide side chain, coumarin ring, and sugarmay be modified in accordance with the other examples shown herein.

Example 71 Coumarin Side Chains

The I analogues are directed to other side-chains extending from thecoumarin ring. As an example, the KU-1/A4 coumarin ring will be preparedfrom 2,4-dihydroxy-5-nitrobenzaldehyde (see Chandrashekhar et al.,g-substitution in the resorcinol nucleus, VI. Formylation of 4-nitro and2-nitro resorcinols, Proc. Ind. Acad. Sci. 1949, 29A, 227-230) and2,4-dihydroxy-5-methoxybenzaldehyde (Demyttenaere et al., Synthesis of6-methoxy-4H-1-benzopyran-7-ol, a character donating component of thefragrance of Wisteria sinensis, Tetrahedron 2002, 58, 2163-2166)according to the procedure of Khoo et al., Synthesis of substituted3-aminocoumarins from ethyl N-2-Hydroxyarylideneglycinates, Syn. Commun.1999, 29, 2533-2538, as generally set forth in the scheme below. Theo-hydroxybenzaldehyde will be treated with ethyl glycine under acidicconditions to afford the corresponding free amine upon basic workup.Both the amino and hydroxyl functionalities will be acylated with thesame anhydrides as shown above. Subsequent hydrolysis of the phenolicester will provide the coumarin amide, which can be coupled directlywith noviose carbonate as described previously.

wherein in the scheme R is hydrogen, alkyl, alkenyl, alkynyl, aryl,carbocylic, heterocyclic, aryl, or aralkyl;

wherein X is alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, halogen ornitro.

Again, while the scheme above illustrates the modified coumarin ring ofKU-1/A4 with a limited number of amide side chain substitutions, it willbe appreciated to those skilled in the art that other derivatives can beprepared in accordance with the above scheme, in addition to the KU-1/A4analogues shown. That is, the amide side chain, coumarin ring, and sugarmay be modified in accordance with the other examples shown herein.

Example 7J Heterocycles

The J analogues will be prepared from4-chloro-2-hydroxy-5-nitrobenzaldehyde (see Pal et al., Newarylsulfonylhydrazones of substituted benzaldehyde as anticancer agents,Neoplasms 1983, 30, 551-556) by treatment with glycine, aceticanhydride, and sodium acetate as mentioned previously for thepreparation of other coumarin derivatives as set forth in the followingscheme. See Khoo et al., Synthesis of substituted 3-aminocoumarins fromethyl N-2-Hydroxyarylideneglycinates, Syn. Commun. 1999, 29, 2533-2538.The chloro substituent will undergo nucleophilic aromatic displacementwith ammonia as a consequence of the electron withdrawing p-lactone ando-nitro group. Upon formation of the 7-amino-6-nitrocoumarin, the nitrogroup will be reduced and immediately treated with triethyl orthoformateto produce the imidazole ring that resembles guanine. See Buckle et al.,Aryloxyalkyloxy- and aralkyloxy-4-hydroxy-3-nitro coumarins whichinhibit histamine release in the rat and also antagonize the effects ofa slow reacting substance of anaphylaxis, J. Med. Chem. 1979, 22,158-168. Subsequent treatment with lithium diisopropylsilylamide andtrimethylsilyl trifluorosulfonic acid will provide the TMS-protecteddiaza compound. See Vorbruggen et al., Organic Reactions, Volume 55,2000, John Wiley and Sons, NY. pp 12-14 and references therein. Thetrichloroacetimidate of noviose carbonate will be added to a solution ofthis TMS-protected coumarin followed by addition of trifluoroacetic acidto afford the coupled product. Upon exposure of the cyclic carbonate totriethylamine in methanol, the resulting diol will be produced in asimilar fashion as was used to make KU-1/A4 directly from thecorresponding cyclic carbonate.

wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocylic,heterocyclic, aryl, or aralkyl.

Example 7K

The K analogues of the KU-1/A4 coumarin moiety will be prepared from5-methoxy-2-methylbenzonitrile as set forth in the scheme below. SeeTomita et al., Schmidt reaction with benzocycloalkenones, J. Chem. Soc.C: Organic 1969, 2, 183-188. Bromination of the benzylic methyl group,followed by displacement with potassium cyanide will furnish thedinitrile product, which is a substrate for acid catalyzed cyclizationto form the corresponding 2-bromoisoquinoline. See Johnson et al., Thecyclization of dinitriles by anhydrous halogen acids. A new synthesis ofisoquinolines, J. Org. Chem. 27, 3953-3958. Acylation of the free aminewith the anhydrides shown in Scheme 4 will furnish the amide products,which will be treated with dilute hydrochloric acid to produce theisoquinolone. As before, the free phenol will be coupled with noviosecarbonate trichloroacetimidate, followed by removal of the cycliccarbonate to furnish K and its acylated (R) derivatives.

wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocylic,heterocyclic, aryl, or aralkyl.

Again, while the scheme above illustrates the modified coumarin ring ofKU-1/A4 with a limited number of amide side chain substitutions, it willbe appreciated to those skilled in the art that other derivatives can beprepared in accordance with the above scheme, in addition to the KU-1/A4analogues shown. That is, the amide side chain, coumarin ring, and sugarmay be modified in accordance with the other examples shown herein.

Example 7L Quinolines

Quinoline derivatives of, L, will be prepared from 7-hydroxyquinoline,by first bromination of the quinoline ring, see Zymalkowski et al.,Chemistry of 3-quinolinecarboxaldehyde, Ann. Chem., Justis Liebigs 1966,699, 98-106, followed by a copper-catalyzed amination of the halogenatedheterocycle as set forth in the scheme below. See Lang et al., Aminationof aryl halides using copper catalysis, Tetrahedron Lett. 2001, 42,4251-3254. Subsequent treatment with various anhydrides (shownpreviously), followed by hydrolysis of the phenolic ester and couplingwith noviose carbonate will ultimately afford these L analogues.

wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocylic,heterocyclic, aryl, or aralkyl.

Again, while the scheme above illustrates the modified coumarin ring ofKU-1/A4 with a limited number of amide side chain substitutions, it willbe appreciated to those skilled in the art that other derivatives can beprepared in accordance with the above scheme, in addition to the KU-1/A4analogues shown. That is, the amide side chain, coumarin ring, and sugarmay be modified in accordance with the other examples shown herein.

Prophetic Example 8 Chlorobiocin Analogues

This example involves the modification of the carbohydrate reside. Morespecifically, analogues similar to that of novobiocin's chlorinatedpyrollic ester, chlorobiocin, will be prepared.

Chlorobiocin

As an example, compound KU-1/A4 will be prepared, and then coupled witha variety of acids to selectively afford the equatorial acylatedalcohols. Selective acylation is based upon previous studies aimed atthe preparation of photolabile derivatives of novobiocin. See Shen etal., Synthesis of Photolabile Novobiocin Analogues, Bioorg. Med. Chem.Lett. 2004, 14, 5903-5906, which is incorporated by reference. Theseacids will include the pyrrolic acid found in chlorobiocin as well asseveral other that are shown in the scheme below. Exemplary acidsinclude pyrrolic acids, indolic acids, pyridinic acids, benzoic acids,salicylic acid, para-hydrobenzoic acid, thiobenzoic acid, and pyrazolicacid. In one aspect, the sugar will be modified to include a functionalgroup according to the formula —R′—OR″, wherein R′ is a covalent bond oralkyl, and R″ is an acyl group. Most preferably, the acyl derivativecomprises the group —COR wherein R is alkyl, aryl, aralkyl, or anaromatic heterocyclic group. Alkylated, aralkylated, thiolated,halogenated, and hydroxylated pyroles, indoles, pyridines, and pyrazolesare attached to the sugar ring as shown in the scheme below.

In another aspect, various substitutents will be added to the amine ofthe carbamate side chain. As an example, carbonate KU-9/A1 will beprepared and amines added to provide the 3′-carbamoyl products asgenerally set forth in the scheme below. Thus, in one aspect the sugarwill be modified to include a functional group according to the formula—R′OR″, wherein R′ is a covalent bond or alkyl, and R″ is C-amido. Mostpreferably, the C-amido group is —CONR′R″ wherein R′ is H, and R″ isalkyl, aryl, aralkyl, or an aromatic heterocyclic group. Pyroles,halogenated benzyls and pyridines, and alkyl groups are shown as themodified side chain of the sugar in the scheme below.

Wherein X is alkyl, alkenyl, alkynyl, hydroxyl, halo, and n is aninteger, preferably 0, 1, 2, 3, or 4.

Prophetic Examples 9-11 Furanose and Pyranose Novobiocin Derivatives

In this example, new pyranose and furanose derivatives will be preparedthat have affinity with the sugar of GTP and phosphate binding region ofHsp90. These selected compounds are shown in below and include ester,amide, sulfonic ester, phosphonic ester, carbamoyl, sulfonamide, andhydroxyl derivatives. Initial compounds will be coupled with thecoumarin ring present in KU-1/A4, but when a more potent analogue isobtained, the best sugar derivative from these studies will be placedonto the optimized ring system.

Examples 9 and 10 Synthesis of Furanose Derivatives

The o-acetyl derivative will be prepared from ribose (9.1, Scheme 9).Treatment of the ribose hemiacetal with benzyl alcohol and hydrochloricgas will provide the benzyloxyacetal, 9.2. See Pigro et al., Readilyavailable carbohydrate-derived imines and amides as chiral ligands forasymmetric catalysis, Tetrahedron 2002, 58, 5459-5466.

Subsequent reaction with carbonyl diimidazole will furnish the2,3-cyclic carbonate (9.3), (See Peixoto et al., Synthesis ofIsothiochroman 2,2-dioxide and 1,2-benzoxathiin 2,2-dioxide Gyrase BInhibitors, Tetrahedron Lett. 2000, 41, 1741-1745) allowing the primaryalcohol to react with acetyl chloride in the following step.Debenzylation, followed by conversion to the trichloroacetimidate 9.5(See Peixoto et al., Synthesis of Isothiochroman 2,2-dioxide and1,2-benzoxathiin 2,2-dioxide Gyrase B Inhibitors, Tetrahedron Lett.2000, 41, 1741-1745) will furnish a suitable substrate for coupling withthe KU-1/A4 coumarin ring system. As noted in previous work, coupling oftrichloroacetimidates with phenols in the presence of catalytic borontrifluoride affords one stereoisomer (9.6), which results from attack ofthe intermediate oxonium species away from the sterically crowded cycliccarbonate. See Shen et al., Synthesis of Photolabile NovobiocinAnalogues, Bioorg. Med. Chem. Lett. 2004, 14, 5903-5906. It has beenpreviously observed that treatment of similar cyclic carbonates withmethanolic triethylamine readily provides the corresponding diolproducts (9.7) in high yields (>80%).

The remaining furanose derivatives will be prepared frombenzyl-protected ribose carbonate (9.3, Scheme 10). Both the sulfonamideand N-acetyl analogues will be furnished by conversion of primaryalcohol (9.3) to the corresponding azide by a Mitsunobu reaction withbis(azido)zinc pyridine complex. Viaud et al., Zinc azide mediatedMitsunobu substitution. An expedient method for the one-pot azidation ofalcohols, Synthesis 1990, 130-132. The resulting azide (10.1) will bereduced, and the primary amine converted to the sulfonamide and N-acetylfunctionalities, 10.2 and 10.3, respectively. Hansson et al., Synthesisof Beta-benzylN-(tert-butoxycarbonyl)-L-erythro-Beta-(benzyloxy)aspartate from(R,R)-(+)-tartaric acid, J. Org. Chem. 1986, 51, 4490-4492. To preparemethyl ester 10.4, the free alcohol will be oxidized directly to theacid, followed by methylation. Carbamate 10.5 will also be prepared fromthe same alcohol, simply by treatment with trichloroacetyl isocyanateaccording to the procedure of Kocovsky, Carbamates: a method ofsynthesis and some synthetic applications, Tetrahedron Lett. 1986, 27,5521-5524. Both the sulfonic ester and the phosphonic ester will beprepared by conversion of 9.3 to iodide 10.6, followed by generation ofthe requisite enolate to displace the halide. Callant et al., Anefficient preparation and the intramolecular cyclopropanation ofBeta-diazo-Beta-ketophosphonates and Beta-diazophosphonoacetates, Syn.Commun. 1984, 14, 155-161. Subsequent treatment with palladium (0) andan amine will lead to allyl removal followed by decarboxylation to form10.10 and 10.8. See Guibe, Allyl esters and their use in complex naturalproduct syntheses, Tetrahedron 1998, 54, 2967-3041.

Example 11 Synthesis of Pyranose Derivatives

The pyranose derivatives, which resemble noviose and a ring-expandedribose ring, will be prepared by our recently reported synthesis of11.1. See Yu et al., Synthesis of Mono- and dihydroxylated furanoses,pyranoses, and an oxepanose for the Preparation of Natural ProductAnalogue Libraries, J. Org. Chem. 2005, 70, 5599-5605, which isincorporated by reference in its entirety. The pyranose derivatives willbe prepared in a similar manner from the known dihydropyrone (See Ahmedet al., Total synthesis of the microtubule stabilizing antitumor agentlaulimalide and some nonnatural analogues: The power of Sharpless'Asymmetric Epoxidation, J. Org. Chem. 2003, 68, 3026-3042), which isavailable in four steps from commercially available triacetyl D-glucal(Roth et al., Synthesis of a chiral synthon for the lactone portion ofcompactin and mevinolin, Tetrahedron Lett. 1988, 29, 1255-12158). Thepyranose will be furnished by Sharpless asymmetric dihydroxylation (SAD)of the olefin to give the product in high diastereomeric excess (Kolb etal., Catalytic Asymmetric Dihydroxylation, Chem. Rev. 1994, 94,2483-2547), which can be converted to the cyclic carbonate at a latertime.

Reduction of the lactone with diisobutylaluminum hydride will givelactol 11.2, which upon treatment with benzyl alcohol and hydrochloricgas will give the benzyloxyacetal 11.3. Similar studies have been usedto prepare noviose from arabinose using an identical sequence of steps.See Peixoto et al., Synthesis of Isothiochroman 2,2-dioxide and1,2-benzoxathiin 2,2-dioxide Gyrase B Inhibitors, Tetrahedron Lett.2000, 41, 1741-1745. The corresponding diol will be treated withcarbonyl diimidazole to yield cyclic carbonate 11.4. The primary alcoholwill be converted to the same functionalities as shown in the schemeabove, using the chemistry depicted for the furanose derivatives.

Once the benzyl protected pyranose derivatives are prepared, they willundergo hydrogenolysis to afford the hemiacetal. Treatment of the lactolwith trichloroacetonitrile will furnish the correspondingtrichloroacetimidate for subsequent coupling with the requisitecoumarin/coumarin analogue. The procedure outlined herein illustratesthe success of coupling such compounds with the coumarin phenol and thisprocedure will be used to prepare the corresponding analogues asdescribed herein.

Using the foregoing schemes, the syntheses of eight protected pyranoseanalogues that include mono- and dihydroxylated variants of bothring-expanded and ring contracted analogues. All eight of thesecompounds were orthogonally protected, such that the hemi-acetal couldbe coupled directly to the coumarin phenol as used similarly for theconstruction of A4. Subsequent removal of the protecting group(s) ortreatment of the cyclic carbonate with ammonia, will afford thecorresponding diol or carbamate products as demonstrated earlier.

Prophetic Example 12 Preparation of 3-Dyhydroxy and 5-DesmethyoylAnalogues

In this example, the 4-deshydroxy and 8-desmethyl variants of novobiocinwill be prepared along with the 8-methyl and 4-hydroxy analogues ofKU-2/A3 (3′carbamate) as shown below. Not only will the 3′-carbamoylderivatives of these compounds be prepared, but also the correspondingdiols for direct comparison to KU-1/A4 (diol).

More specifically, 4-deshydroxynovobiocin will be prepared from3-N-acetyl-7-hydroxy-8-methyl coumarin and the known carboxylic acid asset forth in the scheme below. Spencer et al., Novobiocin. IV. Synthesisof Dihydronovobiocic Acid and Cyclonovobiocic Acid, J. Am. Chem. Soc.1956, 78, 2655-2656. Coupling of these two substrates will provide theamide, which will be treated with noviose carbonate in analogous fashionto other reported syntheses of novobiocin. See Vaterlaus et al., DieSynthese des Novobiocins, Experientia 1963, 19, 383-391; Vaterlaus etal., Novobiocin III Die Glykosidsynthese des Novobiocins, Helv. Chim.Acta 1964, 47, 390-398. Likewise, 8-desmethyl-novobiocin will beprepared from 4,7-dihydroxycoumarin and the diazonium salt to afford themasked amino group similar to our syntheses of photolabile derivatives.See Shen et al., Synthesis of Photolabile Novobiocin Analogues, Bioorg.Med. Chem. Lett. 2004, 14, 5903-5906.

The 7-hydroxyl will undergo selective noviosylation and the diazine willbe reduced. The corresponding amine will be coupled with the knowncarboxylic acid and the carbonate opened with methanolic ammonia to giveboth 3-carbamoyl and diol derivatives.4-Deshydroxy-8-desmethylnovobiocin will be constructed from3-amino-7-hydroxycoumarin in analogous fashion as depicted in the schemebelow. The KU-1/A4 and KU-2/A3 analogues incorporating the same coumarinfunctionalities will be prepared by an identical method (see Khoo,Synthesis of Substituted 3-Aminocoumarins from EthylN-2-Hydroxyarylideneglycinates, Syn. Comm. 1999, 29, 2533-2538) usingacetic anhydride in lieu of the prenylated 4-hydroxybenzoic acid.Des(carbamoyl) derivatives of these compounds will also be prepared byremoval of the cyclic carbonate with triethylamine in methanol, whichaffords similar products in stoichiometric yields.

Prophetic Example 13 Preparation of Dimers

It is contemplated that the C-terminal nucleotide binding sites are inclose proximity to the one another along the Hsp90 dimer interface, andtherefore dimeric inhibitors of the compounds of the present inventionshould provide compounds with enhanced inhibitory activity. This isbased on the fact that the dimeric compound, coumermycin A1, was shownto be approximately 10 times more active than the monomeric compound,novobiocin.

The present invention thus includes dimers of the compounds disclosedherein. In one aspect, a dimeric inhibitor of KU-1/A4 will be prepared.As set forth in the scheme below, the Cbz group will be removed tofurnish the aniline for subsequent coupling with bifunctional linkers toprepare dimeric inhibitors. The dimer containing pyrazole linker foundin Coumermycin A1 will be prepared following the procedure developed byOlson et al., Tetrahedron Letters (2002), Volume Date 2003, 44(1),61-63. The diacid will be coupled with two equivalents of the coumarinamine using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) to furnish the cyclic carbonate precursor tothe KU-1/A4 dimer. The carbonate will be removed upon treatment withmethanolic triethylamine to provide the tetraol product. See Yu et al.Hsp90 Inhibitors Identified from a Library of Novobiocin Analogues. J.Am. Chem. Soc. 127: 12778-12779 (2005). Similar to this method, a numberof dimeric linkers will be used to perturb the dimeric angle and toextend the dimeric tether in an effort to elucidate structure-activityrelationships. As such, ortho, meta, and para dibenzoic acids will beused in lieu of the pyrrole biscarboxylic acid to determine optimalangles. Linker length will be probed by the use of about 3-10 carbondicarboxylic acids. If the studies support that both angle and linkerlength are important, then combinations of these linkers will beprepared and coupled to furnish the conformationally biased, extendedcompounds such as that shown below.

Prophetic Example 14 Prostate Cancer Xenograft Tumor Model

This example involves the in vivo effect of the compounds of the presentinvention using a prostate cancer mouse model. More specifically, fourto six week old BALB/c nu/nu nude mice will be obtained commercially andmaintained in ventilated cages under Institutional Animal Care and UseCommittee approval. Separate male mice will be inoculated subcutaneouslywith 10⁶ LNCaP cells suspended in 0.25 mL of Matrigel (BD, Bioscience,Bedford Mass.). Stable serum testosterone levels will be maintained inthe mice by the implantation of 12.5 mg 90-day sustained releasetestosterone pellets (Innovative Research, Sarasota Fla.) subcutaneouslyprior to inoculation with tumor. Tumor volume will be measured twice aweek with vernier calipers with tumor volumes calculated using theformula [length×width×height×0.52]. Mice with established tumor volumesof 5 mm will be selected for KU-1/A4 administration. Utilizing theparadigm for administration of 17-AAG (another Hsp90 inhibitor), animalswill be treated with both continuous and intermittent dosing schedules.A control animal will be treated with vehicle alone (DMSO). For thecontinuous dosing schedule, mice will receive intraperitoneal injectionsof vehicle or the test compounds (e.g., KU-1/A4) for 5 days per week for3 weeks. The intermittent group will receive one 5 day cycle and thenmonitored for progression.

Differing doses of the test compound (e.g., KU-1/A4) will be utilizedbased on pharmacokinetic information obtained from toxicity studies.When progression occurs, as defined by an increase in tumor size, themice will receive a second 5 day cycle of the test compound (e.g.,KU-1/A4). Response to the test compound will be assessed by measuringtumor volume and serum PSA levels using the PSA Assay Kit (AmericanQualex Antibodies, San Clemente, Calif.). Further response will beassessed by harvesting the tumor at euthanasia and performingimmunohistochemistry and western blot analysis of the Hsp90's clientproteins known to be involved in cancer cell survival mechanisms such assignal transduction (e.g., AKT, Her2, PI3kinase), angiogenesis (e.g.,HIF-1α), and metastasis (AR, MMP2). Each dose and control will berepeated three times to confirm results.

Statistical analysis will be performed to compare the average tumorvolume over time between the different doses of the test compound andthe control animals. The null hypothesis which is that KU-1/A4 willcause no change in tumor volume over time will be tested by the squareddifference between mean tumor volume summed over all time points. Wewill use a Wilcoxon sum-rank test to compare PSA levels in the treatmentand control group. Immunohistochemistry results will be assessedqualitatively based on staining intensity graded on a scale of 1 to 5.

To investigate toxicity, four to six week old BALB/c nu/nu nude micewill be obtained commercially and maintained in ventilated cages underInstitutional Animal Care and Use Committee approval. Intraperitonealinjections of the test compound (e.g., KU-1/A4) will be given tonon-tumor bearing mice at ranges of 25 mg/kg to 200 mg/kg 5 days a weekfor 3 weeks based on similar concentrations used for 17AAG.¹² Serumsamples will be obtained on days 5, 10, and 15. Serum chemistry andliver function analysis will be performed. Serum concentrations of testcompound (e.g., KU-1/A4) will be determined by high performance liquidchromatography (HPLC). At sacrifice by CO₂ euthanasia, a complete bloodcount, gross necropsy and liver and kidney histopathology will beperformed on the animals to determine toxicity. The maximal tolerateddose will be calculated using up/down toxicity studies that will be usedas the upper limit of dose for treatment.

Example 15 Neuroprotective Effects

Recently, low concentrations of the Hsp90 inhibitor GDA were reported toinduce expression of both Hsp70 and Hsp90, with a concomitant reductionin phosphorylated Tau (Dou et al., 2003). In this example, KU-1/A4, anovel C-terminal Hsp90 inhibitor, was tested for protective effectsagainst Aβ toxicity in primary neurons. See protocols in Michaelis M L,Ansar S, Chen Y, Reiff E R, Seyb K I, Himes R H, Audus K L, Georg G I(2005) B-Amyloidinduced neurodegeneration and protection by structurallydiverse microtubule-stabilizing agents. J Pharmacol Exp Ther312:659-668, which is incorporated by reference.

As is shown in FIG. 4, concentrations of KU-1/A4 as low as 5 nMprotected the neurons against Aβ, and the drug alone produced notoxicity. GDA partially protect the neurons against Aβ, but the drugalone was toxic to the neurons at concentrations above 20 nM. Thus,although GDA can increase Hsp90 levels, the result may be thedegradation of client proteins essential for neuronal survival. Thislack of KU1 toxicity in both proliferating and post-mitotic cellssuggested that further exploration of its mechanism(s) of action iswarranted.

From the foregoing it will be seen that this invention is one welladapted to attain all ends and objectives herein-above set forth,together with the other advantages which are obvious and which areinherent to the invention. Since many possible embodiments may be madeof the invention without departing from the scope thereof, it is to beunderstood that all matters herein set forth are to be interpreted asillustrative, and not in a limiting sense. Further, it will beunderstood that certain features and subcombinations are of utility andmay be employed without reference to other features and subcombinations.This is contemplated by and is within the scope of the claims.

1. A compound according to Formula I

wherein R¹ is hydrogen, alkyl, aryl or amido; wherein R² is hydrogen;hydroxy or —R⁸—OR⁹, wherein R⁸ is a covalent bond or alkyl, and R⁹ isC-amido or acyl; or R² together with R³ and the atoms to which they areattached form a heterocyclic ring having 4 to 8 ring members with atleast one heteroatom selected from oxygen or nitrogen; wherein R³ ishydrogen; hydroxy or —R¹⁰—OR¹¹, wherein R¹⁰ is a covalent bond or alkyl,and R¹¹ is C-amido or acyl; or R³ together with R² and the atoms towhich they are attached form a heterocyclic ring having 4 to 8 ringmembers with at least one heteroatom selected from oxygen or nitrogen;wherein R⁴ is hydrogen or methyl; wherein R⁵ is hydrogen or alkyl;wherein R⁶ is hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy,aryloxy, or aralkoxy; wherein X₁ is —O—, —CO—, or —N—; wherein X₂ is—O—, —N—, —NR¹⁸—, —CR¹⁹—, or —CO—; wherein R¹⁸ and R¹⁹ are hydrogen,alkyl, alkenyl, or alkynyl; wherein X₄ is —CR²⁰—, wherein R²⁰ ishydrogen, alkyl, alkenyl, or alkynyl; wherein X₅ is —CR²¹—, wherein R²¹is hydrogen, alkyl, alkenyl, or alkynyl; wherein X₆ is —CR²²—, whereinR²² is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, aralkyl,halogen, or nitro; wherein X₈ is —CR²³— or —N—, wherein R²³ is hydrogen,alkyl, alkenyl, or alkynyl; wherein X₉ is ether; and wherein at leastone of X₁, X₂, X₄, X₅, X₆, X₈ is not —CR—; wherein n is 1; and whereinR² and R³ are not simultaneously hydroxy.
 2. The coumarin compound ofclaim 1 wherein X₁ is —O— and X₂ is —CO—.
 3. The isocoumarin compound ofclaim 1 wherein X₁ is —CO— and X₂ is —O—.
 4. The compound of claim 2wherein R⁴ and R⁵ are both methyl.
 5. The compound of claim 2 wherein X₄is —CR²⁰— and R²⁰ is hydrogen, and wherein X₈ is —CR²³— and R²³ ishydrogen or alkyl.
 6. The compound of claim 5 wherein R¹ is amido. 7.The compound of claim 6 selected from the group consisting of:

N-(7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-2-oxo-2H-chromen-3-yl)acetamide(A1);

(2R,3R,4R,5R)-2-(3-acetamido-2-oxo-2H-chromen-7-yloxy)-4-hydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-3-ylcarbamate (A2);

(3R,4S,5R,6R)-6-(3-acetamido-2-oxo-2H-chromen-7-yloxy)-5-hydroxy-3-methoxy-2,2-dimethyltetrahydro-2H-pyran-4-yl carbamate (A3); and

4-deshydroxy-8-desmethylnovobiocin.
 8. The compound of claim 5 whereinR¹ is hydrogen.
 9. The compound of claim 8 selected from the groupconsisting of:

7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-2H-chromen-2-one (B1);

(3R,4S,5R,6R)-5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-4-ylcarbamate (B2);

(2R,3R,4R,5R)-4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-3-ylcarbamate (B3).
 10. The compound of claim 2 wherein X₄, is —CR²⁰— andR²⁰ is alkyl and wherein R¹ is aryl.
 11. The compound of claim 10selected from the group consisting of:

7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxo-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-4-methyl-3-phenyl-2H-chromen-2-one(C1);

(3R,4S,5R,6R)-5-hydroxy-3-methoxy-2,2-dimethyl-6-(4-methyl-2-oxo-3-phenyl-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-4-ylcarbamate (C2); and

(2R,3R,4R,5R)-4-hydroxy-5-methoxy-6,6-dimethyl-2-(4-methyl-2-oxo-3-phenyl-2H-chromen-7-yloxy)-tetrahydro-2H-pyran-3-ylcarbamate (C3).
 12. The compound of claim 2 wherein X₄ is —CR²⁰— and R²⁰is hydrogen, and wherein X₈ is —CR²³— and R²³ is alkyl.
 13. The compoundof claim 12 wherein R¹ is amido.
 14. The compound of claim 13 selectedfrom the group consisting of:

4-deshydroxynovobiocin (DHN1); and

(3R,4S,5R,6R)-6-(3-acetamido-2-oxo-2H-chromen-7-yloxy)-8-methyl-5-hydroxy-3-methoxy-2,2-dimethyltetrahydro-2H-pyran-4-ylcarbamate (8-methyl A3).
 15. A compound according to Formula II:

wherein R² is hydrogen; hydroxy or —R⁸—OR⁹, wherein R⁸ is a covalentbond or alkyl, and R⁹ is C-amido or acyl; or R² together with R³ and theatoms to which they are attached form a heterocyclic ring having 4 to 8ring members with at least one heteroatom selected from oxygen ornitrogen; and wherein R³ is hydrogen; hydroxyl or —R¹⁰—OR¹¹, wherein R¹⁰is a covalent bond or alkyl, and R¹¹ is C-amido or acyl; or R³ togetherwith R² and the atoms to which they are attached form a heterocyclicring having 4 to 8 ring members with at least one heteroatom selectedfrom oxygen or nitrogen.
 16. The compound of claim 15 selected from thegroup consisting of:

8-(7-Methoxy-6,6-dimethyl-2-oxo-tetrahydro-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-chromen-2-one(D1);

Carbamic acid4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-8-yloxy)-tetrahydro-pyran-3-ylester (D2);

Carbamic acid5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-8-yloxy)-tetrahydro-pyran-4-ylester (D3); and

8-(3,4-Dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-pyran-2-yloxy)-chromen-2-one(D4).
 17. A compound according to Formula III:

wherein R² is hydrogen; hydroxy or —R⁸—OR⁹, wherein R⁸ is a covalentbond or alkyl, and R⁹ is C-amido or acyl; or R² together with R³ and theatoms to which they are attached form a heterocyclic ring having 4 to 8ring members with at least one heteroatom selected from oxygen ornitrogen; and wherein R³ is hydrogen; hydroxyl or —R¹⁰—OR¹¹, wherein R¹⁰is a covalent bond or alkyl, and R¹¹ is C-amido or acyl; or R³ togetherwith R² and the atoms to which they are attached form a heterocyclicring having 4 to 8 ring members with at least one heteroatom selectedfrom oxygen or nitrogen.
 18. The compound of claim 17 selected from thegroup consisting of:

6-(7-Methoxy-6,6-dimethyl-2-oxo-tetrahydro-[1,3]dioxolo[4,5-c]pyran-4-yloxy)-chromen-2-one[E1];

Carbamic acid5-hydroxy-3-methoxy-2,2-dimethyl-6-(2-oxo-2H-chromen-6-yloxy)-tetrahydro-pyran-4-ylester (E2);

Carbamic acid4-hydroxy-5-methoxy-6,6-dimethyl-2-(2-oxo-2H-chromen-6-yloxy)-tetrahydro-pyran-3-ylester (E3); and

6-(3,4-Dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-pyran-2-yloxy)-chromen-2-one(E4).